Methods for improving crispr/cas-mediated genome-editing

ABSTRACT

This application provides improved methods of editing the genome of a target cell. Cas9 molecules can be used to create a break in a genomic region of interest. To increase the likelihood that the break is repaired by homology-directed repair (HDR), the cell can be contacted with an HDR-enhancer. The cell may be, e.g., a human cell, a non-human animal cell, a bacterial cell, or a plant cell.

RELATED APPLICATIONS

This application is a continuation application of U.S. patent application Ser. No. 15/524,672 filed on May 5, 2017, which is a U.S. national stage application, filed under 35 U.S.C. § 371, of International Application No. PCT/US2015/059782, filed on Nov. 9, 2015, which claims the benefit of U.S. Provisional Patent Application No. 62/077,084, filed on Nov. 7, 2014, and of U.S. Provisional Patent Application No. 62/232,683, filed Sep. 25, 2015. The entire contents of each of the foregoing applications are expressly incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XMLcopy, created on May 18, 2023, is named 126454-00104_SL.xml and is 3,113,175 bytes in size.

BACKGROUND

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system evolved in bacteria and archaea as an adaptive immune system to defend against viral attack. Upon exposure to a virus, short segments of viral DNA are integrated into the CRISPR locus. RNA is transcribed from a portion of the CRISPR locus that includes the viral sequence. That RNA, which contains sequence complimentary to the viral genome, mediates targeting of a Cas9 protein to the sequence in the viral genome. The Cas9 protein cleaves and thereby silences the viral target.

Recently, the CRISPR/Cas system has been adapted for genome editing in eukaryotic cells. The introduction of site-specific double strand breaks (DSBs) enables target nucleic acid alteration. After the formation of a DNA double-stranded break (DSB), the major decision point affecting DNA repair pathway choice is whether or not the DNA ends are endo- and exonucleolytically processed in a process referred to as end resection. When no end resection takes places, the repair pathway engaged to repair the DSB is referred to as classical non-homologous end joining (C-NHEJ). The C-NHEJ repair pathway leads to either perfect repair of the DSBs, in which case the locus is restored without sequence alterations, or to the formation of small insertions and deletions.

In contrast, if the end resection machinery processes the DSB, a 3′ overhang is exposed, which engages in homology search. A not yet completely characterized class of pathways that can engage the repair of DSBs after resection is initiated is referred to as alternative non-homologous end joining (ALT-NHEJ). Examples of pathways that are categorized as ALT-NHEJ include blunt end-joining (blunt EJ) and microhomology mediated end joining (MMEJ) leading to deletions, as well as synthesis dependent micro homology mediated end joining (SD-MMEJ), leading to the formation of insertions.

When the end resection is extensive, the exposed 3′ overhang can undergo strand invasion of highly homologous sequences, followed by repair of the DSB by a homology-dependent recombination (HDR) pathway. The HDR pathway comprises homologous recombination (HR), single strand annealing (SSA), and a potential third, not yet fully characterized alternative-HR pathway (“alt-HR”).

While a cell could, in theory, repair breaks via any of a number of DNA damage repair pathways, in certain circumstances it is particularly useful to provide an environment more favorable for repair of the break by a HDR pathway. However, there remains a need to improve the efficiency of HDR-mediated modification in order to broaden the applicability of genome editing by the CRISPR/Cas system.

SUMMARY

This disclosure provides systems and methods for editing a genome using a CRISPR/Cas system which enables target nucleic acid alteration by homology-directed repair (HDR) pathways. In HDR, a cell repairs a damaged region by using a homologous template. In normal cells, this template is often a sister chromatid. To encourage the cell to repair a break by HDR, one can provide an exogenous template nucleic acid that bears, for example, the “correct” sequence corresponding to a mutation. To even further increase the likelihood that the cell repairs the break using HDR, one can contact the cell with an HDR-enhancer. Some HDR-enhancers are agents that inhibit another DNA damage repair pathway, with the result that the cell becomes more likely to use a HDR pathway than the inhibited DNA damage repair pathway. Other HDR-enhancers directly stimulate a HDR pathway. In another embodiment, to encourage the cell to repair a break by HDR, one can optimize the DNA cut. For example, dual gRNAs can be designed to be oriented on a target nucleic acid such that the protospacer adjacent motifs (PAMs) are facing out, and cutting with a Cas9 nickase molecule will result in 5′ overhangs.

In one aspect, described herein is a Cas9 system comprising a gRNA molecule capable of targeting a Cas9 molecule to a target nucleic acid in a cell, a Cas9 molecule, and an HDR-enhancer molecule. In one embodiment, described herein is a Cas9 system comprising an HDR-enhancer molecule and a gRNA molecule. In another embodiment, described herein is a Cas9 system comprising an HDR-enhancer molecule and a Cas9 molecule. In one embodiment, the HDR-enhancer molecule is not an inhibitor of DNA-PK. In one embodiment, the HDR-enhancer molecule is not an inhibitor of Ligase IV.

In one embodiment, the HDR-enhancer molecule is an HDR-enhancing gRNA. In another embodiment, the HDR-enhancer molecule is an siRNA. In another embodiment, the HDR-enhancer molecule is an antibody. In another embodiment, the HDR-enhancer molecule is an miRNA. In another embodiment, the HDR-enhancer molecule is an antiMiR. In another embodiment, the HDR-enhancer molecule is a small molecule. In another embodiment, the HDR-enhancer molecule is a protein. In one embodiment, the protein is a dominant negative protein.

In one embodiment, the HDR-enhancer is a down-regulator of HR, a down-regulator of canonical NHEJ, a down-regulator of alt-NHEJ, a down-regulator of an antirecombinant factor, a down-regulator of SSA, a down-regulator of SSBR, a down-regulator of MMR, a chromatin modification agent, a cell cycle arrest compound, an agent capable of promoting resection at a double strand break, a down-regulator of SD-MMEJ, or a down-regulator of blunt EJ.

In one embodiment, the Cas9 system further comprises a template nucleic acid. In one embodiment, the template nucleic acid is an endogenous nucleic acid in a cell.

In one embodiment, the Cas9 system further comprises a second gRNA suitable for targeting a Cas9 molecule to the target nucleic acid, or a second nucleic acid encoding a second gRNA suitable for targeting a Cas9 molecule to the target nucleic acid. In one embodiment, the Cas9 system further comprises a second gRNA or a second nucleic acid encoding a second gRNA. In one embodiment, the gRNA is configured to guide the Cas9 molecule to produce a first break, and the second gRNA is configured to guide a second Cas9 molecule to produce a second break. In another embodiment, the gRNA and the second gRNA are configured to position the first break and the second break within 65 nucleotides of one another; at least 25 nucleotides apart; or within 25-65 nucleotides of one another.

In another aspect, described herein is a Cas9 system comprising a Cas9 nickase molecule, a gRNA molecule, wherein the gRNA molecule is capable of targeting the Cas9 nickase molecule to a target nucleic acid, and a second gRNA molecule, wherein the second gRNA molecule is capable of targeting the Cas9 nickase molecule to the target nucleic acid, wherein the gRNA molecule and the second gRNA molecule are designed to be oriented on the target nucleic acid such that protospacer adjacent motifs (PAMs) are facing out, wherein the gRNA molecule will position the Cas9 nickase molecule to make a single-stranded break in the target nucleic acid which results a 5′ overhang in the target nucleic acid. In one embodiment, the Cas9 nickase molecule has a D10A mutation. In another embodiment, the gRNA molecule will position the Cas9 nickase molecule to make a single-stranded break in the target nucleic acid which results in a 3′ overhang in the target nucleic acid. In another aspect, described herein is a Cas9 system comprising a Cas9 nickase molecule, a gRNA molecule, wherein the gRNA molecule is capable of targeting the Cas9 nickase molecule to a target nucleic acid, and a second gRNA molecule, wherein the second gRNA molecule is capable of targeting the Cas9 nickase molecule to the target nucleic acid, wherein the gRNA molecule and the second gRNA molecule are designed to be oriented on the target nucleic acid such that protospacer adjacent motifs (PAMs) are facing out, wherein the gRNA molecule will position the Cas9 nickase molecule to make a single-stranded break in the target nucleic acid which results a 3′ overhang in the target nucleic acid. In one embodiment, the Cas9 nickase molecule has an N863A mutation.

In one embodiment, the Cas9 system comprises a gRNA. In one embodiment, the Cas9 system comprises a nucleic acid encoding a gRNA. In one embodiment, the Cas9 system comprises both a gRNA and a nucleic acid encoding a gRNA.

In on embodiment, the Cas9 system comprises a second gRNA. In one embodiment, the Cas9 system comprises a second nucleic acid encoding a second gRNA. In another embodiment, the Cas9 system comprises both a second gRNA and a nucleic acid encoding a second gRNA.

In one embodiment, the Cas9 system comprises a Cas9 polypeptide. In one embodiment, the Cas9 system comprises a nucleic acid encoding a Cas9 polypeptide. In one embodiment, the Cas9 system comprises both a Cas9 polypeptide and a nucleic acid encoding a Cas9 polypeptide.

In one embodiment, the Cas9 system comprises an HDR-enhancer. In one embodiment, the Cas9 system comprises a nucleic acid encoding an HDR-enhancer. In one embodiment, the Cas9 system comprises both an HDR-enhancer and a nucleic acid encoding an HDR-enhancer.

In one embodiment, the Cas9 system comprises a gRNA, a Cas9 polypeptide, and an HDR-enhancer. In another embodiment, the Cas9 system comprises a nucleic acid encoding a gRNA, a nucleic acid encoding a Cas9 polypeptide, and a nucleic acid encoding an HDR-enhancer. In another embodiment, the Cas9 system comprises a nucleic acid encoding a gRNA, a Cas9 polypeptide, and an HDR-enhancer. In another embodiment, the Cas9 system comprises a nucleic acid encoding a gRNA, a nucleic acid encoding a Cas9 polypeptide, and an HDR-enhancer. In another embodiment, the Cas9 system comprises a gRNA, a Cas9 polypeptide, and a nucleic acid encoding an HDR-enhancer. In another embodiment, the composition comprises a gRNA, a nucleic acid encoding a Cas9 polypeptide, and a nucleic acid encoding an HDR-enhancer. In one embodiment, the Cas9 system further comprises a template nucleic acid.

In one embodiment, the HDR-enhancer molecule is an antibody, an miRNA, an siRNA, an antiMiR, a small molecule, or an HDR-enhancing gRNA. In one embodiment, the HDR-enhancer molecule is an miRNA. In one embodiment, the HDR-enhancer molecule is an siRNA. In one embodiment, the HDR-enhancer molecule is an antiMiR. In one embodiment, the HDR-enhancer molecule is an HDR-enhancing gRNA. In one embodiment, the HDR-enhancer molecule is a small molecule. In one embodiment, the HDR-enhancer molecule is an antibody. In one embodiment, the antibody is an intrabody. In one embodiment, the antibody comprises a nuclear localization sequence. In one embodiment, the siRNA is an siRNA comprising a modified nucleotide. In one embodiment, the siRNA is directed against an mRNA that encodes a target.

In one embodiment, the HDR-enhancer molecule is not an inhibitor of RAD51. In one embodiment, the HDR-enhancer molecule is not an inhibitor of BRCA2. In one embodiment, the HDR-enhancer molecule is not an inhibitor of PALB2. In one embodiment, the HDR-enhancer molecule is not an inhibitor of SHFM1. In one embodiment, the HDR-enhancer molecule is not an inhibitor of Ku70. In one embodiment, the HDR-enhancer molecule is not an inhibitor of Ku80. In one embodiment, the HDR-enhancer molecule is not an inhibitor of DNA-PKcs. In one embodiment, the HDR-enhancer molecule is not an inhibitor of XRCC4. In one embodiment, the HDR-enhancer molecule is not an inhibitor of XLF. In one embodiment, the HDR-enhancer molecule is not an inhibitor of Ligase IV. In one embodiment, the HDR-enhancer molecule is not an inhibitor of PNK. In one embodiment, the HDR-enhancer molecule is not an inhibitor of Artemis. In one embodiment, the HDR-enhancer molecule is not PARP1. In one embodiment, the HDR-enhancer molecule is not PARP2. In one embodiment, the HDR-enhancer molecule is not XRCC1. In one embodiment, the HDR-enhancer molecule is not Ligase III. In one embodiment, the HDR-enhancer molecule is not Histone H1.

In one embodiment, the HDR-enhancer molecule is a down-regulator of anti-HR. In one embodiment, the down-regulator of anti-HR is an inhibitor of a protein which inhibits HR or promotes repression of HR. In one embodiment, the down-regulator of anti-HR is capable of promoting SSA or alt-HR. In one embodiment, the down-regulator of anti-HR is capable of promoting SSA or alt-HR as compared to the level of SSA or alt-HR in the absence of the down-regulator of anti-HR.

In one embodiment, the HDR-enhancer molecule is an inhibitor of a component of Table VI.4 or Table VI.1(D). In one embodiment, the HDR-enhancer molecule is an antibody. In one embodiment, the antibody is an intrabody. In one embodiment, the HDR-enhancer molecule is an siRNA. In one embodiment, the HDR-enhancer molecule is an HDR-enhancing gRNA.

In one embodiment, the antibody, the siRNA, or the HDR-enhancing gRNA is directed against a component from Table VI.4 or Table VI.1(D). In one embodiment, the HDR-enhancer molecule is an agent of Table VI.5. In one embodiment, the HDR-enhancer inhibits Fbh1, PARI, RTEL, Rap80, miR-155, miR-545, miR-107, miR-1255, miR-148, or miR-193. In one embodiment, the HDR-enhancer that inhibits Fbh1 is an siRNA. In one embodiment, the HDR enhancer that inhibits PARI is an siRNA. In one embodiment, the HDR-enhancer that inhibits RTEL is an siRNA. In one embodiment, the HDR-enhancer that inhibits RAP80 is an siRNA. In one embodiment, the HDR-enhancer that inhibits miR-155, miR-545, miR-107, miR-1255, miR-148, or miR-193 is an anti-miR. In another embodiment, the HDR-enhancer is an HDR-enhancing gRNA molecule.

In one embodiment, the HDR-enhancer molecule is a down-regulator of SSA. In one embodiment, the down-regulator of SSA is an inhibitor of a protein, wherein the protein promotes SSA. In one embodiment, the down-regulator of SSA is capable of promoting HR or alt-HR. In one embodiment, the down-regulator of SSA is capable of promoting HR or alt-HR as compared to the level of HR or alt-HR that would occur in the absence of the down-regulator of SSA.

In one embodiment, the HDR-enhancer molecule is an inhibitor of a component of Table VI.1(E) or VI.11. In one embodiment, the HDR-enhancer molecule is an antibody, an siRNA, a small molecule, or an HDR-enhancing gRNA. In one embodiment, the antibody is an intrabody.

In one embodiment, the antibody, the siRNA, or the HDR-enhancing gRNA is directed against a component from Table VI.1(E) or VI.11. In one embodiment, the HDR-enhancer molecule is an agent of Table VI.12.

In one embodiment, the HDR-enhancer molecule inhibits Rad52, XPF, or ERCC1. In one embodiment, the HDR-enhancer molecule that inhibits Rad52, XPF or ERCC1 is an siRNA or an HDR-enhancing gRNA molecule.

In one embodiment, the HDR-enhancer molecule is a chromatin modification agent. In one embodiment, the chromatin modification agent is an agent that inhibits a chromatin modification protein that promotes a DNA repair pathway. In one embodiment, the chromatin modification agent is capable of promoting HDR. In one embodiment, HDR is increased as compared to the level of HDR that would occur in the absence of the chromatin modification agent.

In one embodiment, the chromatin modification agent is not an HDAC. In another embodiment, the HDR-enhancer molecule is an HDAC. In one embodiment, the HDAC is HDAC1 or HDAC2. In one embodiment, the HDR-enhancer that inhibits the HDAC is TCA.

In one embodiment, the HDR-enhancer molecule is present in an amount sufficient to alter chromatin at a target nucleic acid. In one embodiment, the HDR-enhancer molecule is a modulator of a component of Table VI.1(I). In one embodiment, the modulator is an inhibitor. In one embodiment, the HDR-enhancer molecule is an antibody, an siRNA, or a HDR-enhancing gRNA. In one embodiment, the antibody is an intrabody. In one embodiment, the HDR-enhancer molecule is directed against a component from Table VI.1(I). In one embodiment, the HDR-enhancer molecule is an agent of Table VII. In one embodiment, the HDR-enhancer inhibits EZH2. In one embodiment, the HDR-enhancer that inhibits EZH2 is EPZ-6438. In one embodiment, the HDR-enhancer is directed against a component from Table VI.1(I). In one embodiment, the HDR-enhancer is an agent of Table VII. In one embodiment, the HDR-enhancer inhibits Setd2.

In one embodiment, the HDR-enhancer molecule is a down-regulator of SSBR. In one embodiment, the down-regulator of SSBR is an inhibitor of a protein, wherein the protein promotes SSBR. In one embodiment, HDR is increased as compared to the level of HDR that would occur in the absence of the down-regulator of SSBR.

In one embodiment, the HDR-enhancer molecule is an antibody, an siRNA, a small molecule, or an HDR-enhancing gRNA. In one embodiment, the antibody is an intrabody. In one embodiment, the HDR-enhancer molecule is an inhibitor of a component of Table VI.13 or VI.1(F). In one embodiment, the antibody, the siRNA, or the HDR-enhancing gRNA is directed against a component from Table VI.13 or VI.1(F). In one embodiment, the HDR-enhancer molecule is an agent of Table VI.14. In one embodiment, the HDR-enhancer inhibits a PARP or XRCC1. In one embodiment, the HDR-enhancer that inhibits a PARP is selected from the group consisting of AZD2281, KU-0059436, and BMN673. In one embodiment, the HDR-enhancer that inhibits XRCC1 is an siRNA.

In one embodiment, the HDR-enhancer molecule is an agent capable of promoting resection at a single or double strand break. In one embodiment, the agent capable of promoting resection is increases HDR as compared to the level of HDR that would occur in the absence of the agent capable of promoting resection. In one embodiment, the agent that promotes resection at a single or double strand break is an endonuclease or an exonuclease. In one embodiment, the agent that promotes resection is an inhibitor of an anti-resection protein.

In one embodiment, the HDR-enhancer molecule is an antibody, an siRNA, a small molecule, a polypeptide, or an HDR-enhancing gRNA. In one embodiment, the antibody is an intrabody. In one embodiment, the anti-resection protein is a protein of Table VI.1(A). In one embodiment, the antibody, the siRNA, or the HDR-enhancing gRNA is directed against a component from Table VI.1(A). In one embodiment, the inhibitor of an anti-resection protein is an inhibitor of 53BP1, Rif-1, or PTIP. In one embodiment, the inhibitor of an anti-resection protein is a dominant negative 53BP1 protein.

In one embodiment, the HDR-enhancer molecule is a down-regulator of SD-MMEJ. In one embodiment, the HDR-enhancer molecule is an antibody, an siRNA, or a an HDR-enhancing gRNA. In one embodiment, the antibody is an intrabody. In one embodiment, the HDR-enhancer molecule is an inhibitor of Pol Theta. In one embodiment, the HDR-enhancer molecule is a CDK1 inhibitor. In one embodiment, the HDR-enhancer molecule is an agent of Table VIII. In one embodiment, the antibody, the siRNA, or the HDR-enhancing gRNA is directed against a component from Table VIII.

In one embodiment, the HDR-enhancer molecule is an agent that promotes cell cycle arrest in G2 phase, wherein the HDR-enhancer molecule is not a CDK1 inhibitor. In one embodiment, the HDR-enhancer molecule is present in an amount sufficient to cause a cell to arrest in G2 phase.

In one embodiment, the Cas9 system further comprises an additional one or more HDR-enhancer molecules. In one embodiment, the Cas9 system further comprises an additional one HDR enhancer molecule. In one embodiment, the Cas9 system further comprises an additional two HDR-enhancer molecules.

In one embodiment, the HDR-enhancer molecule and the additional one or more HDR-enhancer molecules are capable of up-regulating the same pathway. In one embodiment, the HDR-enhancer molecule and the additional one or more HDR-enhancer molecules are capable of down-regulating the same pathway.

In one embodiment, the HDR-enhancer molecule is a down-regulator of C-NHEJ. In one embodiment, the down-regulator of C-NHEJ is capable of increasing levels of HDR as compared to the level of HDR that would occur in the absence of the down-regulator of C-NHEJ. In one embodiment, the HDR-enhancer molecule is an antibody, an siRNA, a small molecule, or an HDR-enhancing gRNA.

In one embodiment, the HDR-enhancer molecule is an inhibitor of a component of Table VI.7 or VI.1(B). In one embodiment, the antibody, the siRNA, or the HDR-enhancing gRNA is directed against a component from Table VI.7 or VI.1(B). In one embodiment, the HDR-enhancer molecule is an agent of Table VI.8. In one embodiment, the HDR-enhancer molecule is an inhibitor of DNA Pk or an inhibitor of 53BP1. In one embodiment, the inhibitor of DNA Pk is selected from the group consisting of NU7441, KU-0060648, CC115, and NK314. In one embodiment, the inhibitor of 53BP1 is an siRNA targeting 53BP1. In one embodiment, the inhibitor of an anti-resection protein is a dominant negative 53BP1 protein.

In one embodiment, the HDR-enhancer molecule is a down-regulator of blunt EJ. In one embodiment, the HDR-enhancer molecule is a down-regulator of SD-MMEJ. In one embodiment, the down-regulator of blunt EJ or MMEJ is capable of increasing the level of HDR as compared to the level of HDR that would occur in the absence of the down-regulator of blunt EJ or SD-MMEJ. In one embodiment, the HDR-enhancer molecule is an antibody, an siRNA, a small molecule, or an HDR-enhancing gRNA.

In one embodiment, the HDR-enhancer molecule is an inhibitor of a component of Table VI.9 or Table VI.1(J). In one embodiment, the antibody, the siRNA, or the HDR-enhancing gRNA is directed against a component from Table VI.9 or Table VI.1(J). In one embodiment, the HDR-enhancer molecule is an agent of Table VI.10.

In one embodiment, the HDR-enhancer molecule is an up-regulator of HDR. In one embodiment, the up-regulator of HDR is a protein selected from the group consisting of: MRE11, RAD50, NBS1, BRCA2, and BRCA1, or a polypeptide comprising at least 60, 70, 80, 90, 95, 98, 99 or 100% homology with, or which differing by no more than 50, 40, 30, 20, 15, 10, 5, 4, 3, 2, or 1, amino acid residues from a naturally occurring MRE11, RAD50, NBS1, BRCA2, or BRCA1. In one embodiment, the up-regulator of HDR is a protein of Table VI.2 or a protein of Table VI.1(C). In one embodiment, the up-regulator of HDR is a polypeptide comprising at least 60, 70, 80, 90, 95, 98, 99 or 100% homology with, or which differing by no more than 50, 40, 30, 20, 15, 10, 5, 4, 3, 2, or 1, amino acid residues from, a protein of Table VI.2 or Table VI.1(C). In another embodiment, the up-regulator of HDR is a dominant negative CtIP. A dominant negative CtIP promotes resection in G1 phase.

In another embodiment, the HDR-enhancer molecule is an up-regulator of SSA. In one embodiment, the up-regulator of SSA is a protein selected from the group consisting of Rad52 and ERCC1. In one embodiment, the up-regulator of SSA is a polypeptide comprising at least 60, 70, 80, 90, 95, 98, 99 or 100% homology with, or which differing by no more than 50, 40, 30, 20, 15, 10, 5, 4, 3, 2, or 1, amino acid residues from, Rad52 or ERCC1. In one embodiment, the HDR-enhancer molecule is a down-regulator of one HDR pathway. In one embodiment, the down-regulator one HDR Pathway is an inhibitor of a protein, wherein the protein promotes other HDR pathways. In one embodiment, the down-regulator of HDR is capable of increasing alt-HR and/or SSA as compared to the level of alt-HR and/or SSA in the absence of the down-regulator of HDR. In one embodiment, the down-regulator of HDR is capable of increasing alt-HR and/or HR as compared to the level of alt-HR and/or HR in the absence of the down-regulator of HDR. In one embodiment, the down-regulator of HDR is capable of increasing HR and/or SSA as compared to the level of HR and/or SSA in the absence of the down-regulator of −HDR.

In one embodiment, the HDR-enhancer molecule is an inhibitor of a component of Table VI.2 or VI.1(C). In one embodiment, the HDR-enhancer molecule is an antibody, an siRNA, a small molecule, or an HDR-enhancing gRNA. In one embodiment, the antibody is an intrabody. In one embodiment, the antibody, the siRNA, the small molecule, or the HDR-enhancing gRNA is directed against a component from Table VI.2 or VI.1(C). In one embodiment, the HDR-enhancer molecule is an agent of Table VI.3.

In one embodiment, the HDR-enhancer inhibits BRCA2, BRCA1, or RAD51. In one embodiment, the HDR-enhancer molecule is an antibody directed against a BRCA2, BRCA1, or RAD51. In one embodiment, the antibody is an intrabody. In one embodiment, the HDR-enhancer molecule is an siRNA directed against BRCA2, BRCA1, or RAD51. In one embodiment, the HDR-enhancer molecule is selected from: B02, A03, AI-10, RI-1, RI-2, and IBR2.

In one embodiment, the HDR-enhancer molecule is a cell cycle arrest agent. In one embodiment, the Cas9 system of further comprises a cell cycle arrest agent. In one embodiment, the cell cycle arrest agent is capable of arresting cells in G2 phase. In one embodiment, the cell cycle arrest agent is a Cdk1 inhibitor. In one embodiment, the Cdk1 inhibitor is an siRNA or an antibody. In one embodiment, the cell cycle arrest agent is not a Cdk1 inhibitor.

In one embodiment, the gRNA is configured to position a Cas9 molecule-mediated cleavage event at a preselected position relative to a landmark on a target nucleic acid, wherein the target nucleic acid is an endogenous nucleic acid. In one embodiment, the landmark is a preselected site in the target nucleic acid. In another embodiment, the preselected position, or the landmark, or both the preselected position and the landmark, are present on the endogenous nucleic acid. In one embodiment, the endogenous nucleic acid is a chromosomal nucleic acid or an organellar nucleic acid. In one embodiment, the endogenous nucleic acid is not a heterologous reporter gene.

In one embodiment, the gRNA comprises at least one domain of a preselected length. In another embodiment, the at least one domain is a targeting domain.

In one embodiment, the landmark is a target position, wherein the target position is the nucleotide or one of the nucleotides to be corrected or altered. In one embodiment, the landmark is the 5′ end of a target position, wherein the target position is a nucleotide or one of the nucleotides to be corrected or altered. In one embodiment, the landmark is the 3′ end of a target position, wherein the target position is the nucleotide or one of the nucleotides to be corrected or altered. In one embodiment, the landmark is within a target position, wherein the target position is the nucleotide or one of the nucleotides to be corrected or altered.

In one embodiment, the Cas9 system further comprises a template nucleic acid comprising a 5′ homology arm, a replacement sequence, and a 3′ homology arm, wherein the landmark is a position on the target nucleic acid that corresponds to the 5′ end of the replacement sequence.

In one embodiment, the Cas9 system further comprises a template nucleic acid comprising a 5′ homology arm, a replacement sequence, and a 3′ homology arm, wherein the landmark is a position on the target nucleic acid that corresponds to the 3′ end of the replacement sequence.

In one embodiment, the Cas9 system further comprises a template nucleic acid comprising a 5′ homology arm, a replacement sequence, and a 3′ homology arm, wherein the landmark is a position on the target nucleic acid within the replacement sequence.

In one embodiment, the Cas9 system further comprises a template nucleic acid comprising a 5′ homology arm, a replacement sequence, and a 3′ homology arm, wherein the landmark is a position on the target nucleic acid within the 5′ homology arm.

In one embodiment, the Cas9 system further comprises a template nucleic acid comprising a 5′ homology arm, a replacement sequence, and a 3′ homology arm, wherein the landmark is a position on the target nucleic acid within the 3′ homology arm.

In one embodiment, the Cas9 system further comprises a template nucleic acid comprising a 5′ homology arm, a replacement sequence, and a 3′ homology arm, wherein the landmark is a position on the target nucleic acid that corresponds to the 5′ end of the template nucleic acid.

In one embodiment, the Cas9 system further comprises a template nucleic acid comprising a 5′ homology arm, a replacement sequence, and a 3′ homology arm, wherein the landmark is a position on the target nucleic acid that corresponds to the 3′ end of the template nucleic acid.

In one embodiment, the landmark is an intron/exon boundary. In one embodiment, the intron/exon boundary is the intron/exon boundary nearest a target position, wherein the target position is a nucleotide or one of the nucleotides to be corrected or altered. In one embodiment, the intron/exon boundary is within 50, 100, 200, or 500 nucleotides of the target position, wherein the target position is a nucleotide or one of the nucleotides to be corrected or altered.

In one embodiment, the landmark is in an intron. In one embodiment, the intron is the intron nearest to a target position, wherein the target position is a nucleotide or one of the nucleotides to be corrected or altered. In one embodiment, the intron is the nearest intron upstream of a target position, wherein the target position is a nucleotide or one of the nucleotides to be corrected or altered. In one embodiment, the intron is the nearest intron downstream of a target position, wherein the target position is a nucleotide or one of the nucleotides to be corrected or altered. In one embodiment, the intron is an intron within 50, 100, 200, or 500 nucleotides of the target position.

In one embodiment, the landmark is in an exon. In one embodiment, the exon is the exon nearest to a target position, wherein the target position is a nucleotide or one of the nucleotides to be corrected or altered. In one embodiment, the exon is the nearest exon upstream of a target position, wherein the target position is a nucleotide or one of the nucleotides to be corrected or altered. In one embodiment, the exon is the nearest exon downstream of a target position, wherein the target position is a nucleotide or one of the nucleotides to be corrected or altered. In one embodiment, the exon is an exon within 50, 100, 200, or 500 nucleotides of the target position.

In one embodiment, the landmark is the 5′ end of a coding region or the 3′ end of a coding region. In one embodiment, the coding region is the coding region nearest to a target position. In one embodiment, the coding region is the coding region within a target position lies. In one embodiment, the coding region is the nearest coding region downstream of a target position. In one embodiment, the coding region is the nearest coding region upstream of a target position. In one embodiment, the coding region is a coding region within 50, 100, 200, or 500 nucleotides of a target position. In one embodiment, the landmark is within a coding region. In one embodiment, the coding region is the coding region nearest to a target position. In one embodiment, the coding region is the coding region within which a target position lies. In one embodiment, the coding region is the nearest coding region upstream of a target position, or the nearest coding region downstream of a target position. In one embodiment, the coding region is a coding region within 50, 100, 200, or 500 nucleotides of a target position.

In one embodiment, the landmark is the 5′ end of a transcribed region. In one embodiment, the transcribed region is a transcribed region nearest to a target position, a transcribed region within which a target position lies, a nearest transcribed region upstream of a target position, a nearest transcribed region downstream of a target position, or a transcribed region within 50, 100, 200, or 500 nucleotides of a target position.

In one embodiment, the landmark is the 3′ end of a transcribed region. In one embodiment, the transcribed region is a transcribed region nearest to a target position, a transcribed region within which a target position lies, a nearest transcribed region upstream of a target position, a nearest transcribed region downstream of a target position, or a transcribed region within 50, 100, 200, or 500 nucleotides of a target position.

In one embodiment, the landmark is within a transcribed region. In one embodiment, the transcribed region is a transcribed region nearest to a target position, a transcribed region within which a target position lies, a nearest transcribed region upstream of a target position, a nearest transcribed region downstream of a target position, or a transcribed region within 50, 100, 200, or 500 nucleotides of a target position.

In one embodiment, the landmark is the 5′ end of a repeated element. In one embodiment, the landmark is the 3′ end of a repeated element. In one embodiment, the landmark is within a repeated element. In one embodiment, the repeated element is a repeated element nearest to a target position, a repeated element within which a target position lies, a nearest repeated element upstream of a target position, a nearest repeated element downstream of a target position, or a repeated element within 50, 100, 200, or 500 nucleotides of a target position. In one embodiment, the preselected position is at the landmark, not at the landmark, within 50, 100, 150, or 200 nucleotides of the landmark, at least 10, 20, 30, 40, or 50 nucleotides away from the landmark, or 10-200, 20-200, 30-200, 40-200, 50-200, 10-150, 10-100, or 10-50 nucleotides away from the landmark.

In one embodiment, the landmark is a target position, wherein the target position is a nucleotide or one of the nucleotides to be corrected or altered, and the preselected position is at the landmark, away from the landmark, within 50, 100, 150, or 200 nucleotides of the landmark, or 10-200, 20-200, 30-200, 40-200, 50-200, 10-150, 10-100, or 10-50 nucleotides away from the landmark.

In one embodiment, the landmark is a repetitive sequence, and wherein the preselected position is away from the landmark; at least 50, 100, 150, or 200 nucleotides away from the landmark; or 10-200, 20-200, 30-200, 40-200, 50-200, 10-150, 10-100, or 10-50 nucleotides away from the landmark.

In one embodiment, the targeting domain is 12-30 nucleotides in length. In one embodiment, the targeting domain is at least 21 nucleotides in length.

In one embodiment, the at least one domain of a preselected length is a domain encompassing a proximal domain and a tail domain which, taken together, are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides in length.

In one embodiment, the 5′ homology arm has a length of at least 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides. In one embodiment, the 5′ homology arm has a length of no more than 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides. In one embodiment, the 5′ homology arm has a length of between 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides.

In one embodiment, the 5′ homology arm has a length of at least 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides. In one embodiment, the 5′ homology arm has a length of no more than 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides. In one embodiment, the 5′ homology arm has a length of between 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides.

In one embodiment, the 5′ homology arm has a 5′ end and a 3′ end and: the 5′ end is at least 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides from a target position, the 5′ end is no more than 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides from a target position, the 5′ end is between 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides from a target position, the 3′ end is at least 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides from a target position, the 3′ end is no more than 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides from a target position, or the 3′ end is between 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides from a target position.

In one embodiment, the 3′ homology arm has a 5′ end and a 3′ end and: the 5′ end is at least 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides from a target position, the 5′ end is no more than 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides from a target position, the 5′ end is between 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides from a target position, the 3′ end is at least 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides from a target position, the 3′ end is no more than 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides from a target position, or the 3′ end is between 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides from a target position.

In one embodiment, the replacement sequence has a length of: at least 1, 2, 3, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 300, 4000, or 5000 nucleotides, no more than 2, 3, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 300, 4000, or 5000 nucleotides, or between 1-3, 1-5, 1-10 10-20, 20-50, 50-100, 100-200, 200-500, 500-1000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides.

In one embodiment, the gRNA is chimeric. In one embodiment, the gRNA is modular. In one embodiment, the gRNA comprises a targeting domain, a first complementary domain, a second complementary domain, and a proximal domain.

In one embodiment, the template nucleic acid comprises a 5′ homology arm, a replacement sequence, and a 3′ homology arm. In one embodiment, the replacement sequence corresponds to a second endogenous nucleic acid. In one embodiment, the second endogenous nucleic acid is a second chromosomal nucleic acid or a second organellar nucleic acid. In another embodiment, the second endogenous nucleic acid is not a heterologous reporter gene.

In one embodiment, the template nucleic acid comprises, or comprises a part of, a circular nucleic acid. In one embodiment, the circular nucleic acid is a plasmid. In one embodiment, the template nucleic acid is a linear nucleic acid. In one embodiment, the template nucleic acid comprises a double stranded sequence. In one embodiment, the template nucleic acid comprises a single strand oligonucleotide. In one embodiment, the template nucleic acid comprises a single-stranded DNA hybrid. In one embodiment, the template nucleic acid is present in an AAV or an ILDV. In one embodiment, the template nucleic acid is an endogenous nucleic acid sequence.

In one embodiment, the template nucleic acid comprises about 150-200 nucleotides of homology with a target nucleic acid. In one embodiment, the 150-200 nucleotides of homology correspond to one side of a break in the target nucleic acid. In one embodiment, the 150-200 nucleotides of homology correspond to two sides of a break in the target nucleic acid. In one embodiment, the template nucleic acid comprises about 500-2000 nucleotides of homology with a target nucleic acid. In one embodiment, the 500-2000 nucleotides of homology correspond to one side of a break in the target nucleic acid. In one embodiment, the 500-2000 nucleotides of homology correspond to two sides of a break in the target nucleic acid.

In one embodiment, the template nucleic acid has homology to the target nucleic acid on one side of the break. In one embodiment, the template nucleic acid has homology to the target nucleic acid on two sides of the break. In one embodiment, the template nucleic acid comprises a human sequence. In one embodiment, the human sequence is a wild-type human sequence. In one embodiment, the wild-type human sequence corresponds to a mutation at the target nucleic acid. In one embodiment, the template nucleic acid lacks a repeated element. In one embodiment, the repeated element is an Alu sequence or a LINE sequence. In one embodiment, the template nucleic acid comprises a modified nucleic acid.

In one embodiment, the Cas9 molecule is a protein selected from Table 111.1. In another embodiment, the Cas9 molecule is not a S. pyogenes Cas9 molecule. In one embodiment, the Cas9 molecule is a S. pyogenes Cas9 molecule. In another embodiment, the Cas9 molecule is an S. aureus Cas9 molecule. In one embodiment, the Cas9 molecule comprises at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% homology with, or which differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 35, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350 or 400, amino acid residues from, an amino acid sequence of a naturally occurring Cas9 molecule. In one embodiment, the naturally occurring Cas9 molecule is a Cas9 molecule described in Table II.1 herein.

In one embodiment, the Cas9 molecule is less than about 1300 amino acids in length. In another embodiment, the Cas9 molecule comprises a heterologous PI domain. In another embodiment, the Cas9 molecule comprises a REC2 deletion, REC1_(CT) deletion, or a REC1_(SUB) deletion, or any combination thereof.

In one embodiment, the Cas9 system further comprises a second Cas9 molecule. In one embodiment, the Cas9 molecule is a nickase, and the second Cas9 molecule is a nickase. In one embodiment, the Cas9 molecule can catalyze a double strand break, and the second Cas9 molecule is a nickase. In one embodiment, the Cas9 molecule is a nickase, and the second Cas9 molecule can catalyze a double strand break. In one embodiment, the Cas9 molecule can catalyze a double strand break, and the second Cas9 molecule can catalyze a double strand break. In one embodiment, the Cas9 molecule and the second Cas9 molecule have the same amino acid sequence, or wherein the Cas9 molecule and the second Cas9 molecule have different amino acid sequences.

In one embodiment, the Cas9 molecule is an eiCas9 molecule.

In one embodiment, the Cas9 molecule is an eaCas9 molecule. In one embodiment, the eaCas9 can catalyze a double strand break in the target nucleic acid. In one embodiment, the eaCas9 molecule comprises N-terminal RuvC-like domain cleavage activity and HNH-like domain cleavage activity. In one embodiment, the eaCas9 molecule can catalyze a single strand break in a target nucleic acid. In one embodiment, the eaCas9 molecule comprises HNH-like domain cleavage activity but has no, or no significant, N-terminal RuvC-like domain cleavage activity. In one embodiment, the eaCas9 molecule is an HNH-like domain nickase. In one embodiment, the eaCas9 molecule comprises a mutation at D10. In one embodiment, the eaCas9 molecule comprises N-terminal RuvC-like domain cleavage activity but has no, or no significant, HNH-like domain cleavage activity. In one embodiment, the eaCas9 molecule is an N-terminal RuvC-like domain nickase. In one embodiment, the eaCas9 molecule comprises a mutation at H840 or N863.

In one embodiment, the Cas9 molecule is less than about 1200, 1100, 1000, 900, or 800 amino acids in length; or between about 800-1300, 900-1200, 900-1100, or 900-1000 amino acids in length.

In one embodiment, the Cas9 recognizes a PAM site other than NGG, a PAM site other than AGG, or an inverted PAM site.

In one embodiment, described herein is a cell comprising a Cas9 system. In one embodiment, the cell is a eukaryotic cell. In another embodiment, the cell is a vertebrate, mammalian, rodent, goat, pig, bird, chicken, turkey, cow, horse, sheep, fish, primate, or human cell. In another embodiment, the cell is a plant cell. In one embodiment, the plant cell is a monocot or a dicot. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a somatic cell, a germ cell, or a prenatal cell. In one embodiment, the cell is a zygotic cell, a blastocyst cell, an embryonic cell, a stem cell, a mitotically competent cell, or a meiotically competent cell. In one embodiment, the cell is not part of a human embryo. In one embodiment, the cell is a somatic cell. In one embodiment, the cell is a T cell, a CD8+ T cell, a CD8+ naïve T cell, a central memory T cell, an effector memory T cell, a CD4+ T cell, a stem cell memory T cell, a helper T cell, a regulatory T cell, a cytotoxic T cell, a natural killer T cell, a Hematopoietic Stem Cell, a long term hematopoietic stem cell, a short term hematopoietic stem cell, a multipotent progenitor cell, a lineage restricted progenitor cell, a lymphoid progenitor cell, a myeloid progenitor cell, a common myeloid progenitor cell, an erythroid progenitor cell, a megakaryocyte erythroid progenitor cell, a retinal cell, a photoreceptor cell, a rod cell, a cone cell, a retinal pigmented epithelium cell, a trabecular meshwork cell, a cochlear hair cell, an outer hair cell, an inner hair cell, a pulmonary epithelial cell, a bronchial epithelial cell, an alveolar epithelial cell, a pulmonary epithelial progenitor cell, a striated muscle cell, a cardiac muscle cell, a muscle satellite cell, a neuron, a neuronal stem cell, a mesenchymal stem cell, an induced pluripotent stem (iPS) cell, an embryonic stem cell, a monocyte, a megakaryocyte, a neutrophil, an eosinophil, a basophil, a mast cell, a reticulocyte, a B cell, e.g., a progenitor B cell, a Pre B cell, a Pro B cell, a memory B cell, a plasma B cell, a gastrointestinal epithelial cell, a biliary epithelial cell, a pancreatic ductal epithelial cell, an intestinal stem cell, a hepatocyte, a liver stellate cell, a Kupffer cell, an osteoblast, an osteoclast, an adipocyte, a preadipocyte, a pancreatic islet cell (e.g., a beta cell, an alpha cell, a delta cell), a pancreatic exocrine cell, a Schwann cell, or an oligodendrocyte. In one embodiment, the cell is a T cell, a Hematopoietic Stem Cell, a retinal cell, a cochlear hair cell, a pulmonary epithelial cell, a muscle cell, a neuron, a mesenchymal stem cell, an induced pluripotent stem (iPS) cell, or an embryonic stem cell.

In one embodiment, described herein is a composition comprising a Cas9 system. In one embodiment, the composition further comprises a pharmaceutically acceptable excipient. In one embodiment, the composition comprises a cell described herein, or a population of cells comprising cells described herein. In one embodiment, when the composition comprises a gRNA molecule and an HDR-enhancer molecule, the gRNA molecule and the HDR-enhancer molecule form part of a single admixture or are provided separately. In another embodiment, when the composition comprises a Cas9 molecule and an HDR-enhancer molecule, the HDR-enhancer molecule and the Cas9 molecule form part of a single admixture or are provided separately. In another embodiment, when the composition comprises a gRNA molecule, a Cas9 molecule, and an HDR-enhancer molecule; the gRNA molecule, the Cas9 molecule, and the HDR-enhancer molecule form part of a single admixture or are provided separately. In another embodiment, when the composition comprises a gRNA molecule, an HDR-enhancer molecule, and a template nucleic acid; the gRNA molecule, the HDR-enhancer molecule, and the template nucleic acid form part of a single admixture or are provided separately. In another embodiment, when the composition comprises a Cas9 molecule, an HDR-enhancer molecule, and a template nucleic acid; the Cas9 molecule, the HDR-enhancer molecule, and the template nucleic acid form part of a single admixture or are provided separately. In another embodiment, when the composition comprises a gRNA molecule, a Cas9 molecule, an HDR-enhancer molecule, and a template nucleic acid; the gRNA molecule, the Cas9 molecule, the HDR-enhancer molecule, and the template nucleic acid form part of a single admixture or are provided separately.

In one embodiment, described herein is a kit comprising a Cas9 system. In one embodiment, the kit further comprises packaging. In one embodiment, the kit further comprises instructions for use to treat a disorder. In one embodiment, the disorder is a disorder caused by a target position in a target nucleic acid.

In one embodiment, the nucleic acid encoding the gRNA suitable for targeting the Cas9 molecule to the target nucleic acid in the cell is a DNA molecule. In one embodiment, the nucleic acid encoding the Cas9 molecule is a DNA molecule. In one embodiment, the nucleic acid that encodes the HDR-enhancer molecule is a DNA molecule. In one embodiment, the template nucleic acid is a DNA molecule.

In one embodiment, the Cas9 system further comprises a nucleic acid that encodes a second gRNA. In one embodiment, the Cas9 system further comprises a nucleic acid that encodes a second Cas9 molecule. In one embodiment, the Cas9 system further comprises a nucleic acid that encodes a second HDR-enhancer. In one embodiment, the Cas9 system further comprises a nucleic acid that encodes a third HDR-enhancer.

In one embodiment, the HDR-enhancer molecule is a protein. In one embodiment, the HDR-enhancer molecule is an RNA molecule. In one embodiment, the nucleic acid that encodes the HDR-enhancer molecule is a DNA molecule.

In one embodiment, the nucleic acid encoding the gRNA and the nucleic acid encoding the Cas9 polypeptide are present on a single nucleic acid molecule. In another embodiment, the nucleic acid encoding the gRNA and the nucleic acid encoding the Cas9 polypeptide are present on separate nucleic acid molecules.

In one embodiment, the nucleic acid encoding the gRNA and the nucleic acid encoding the HDR-enhancer are present on a single nucleic acid molecule. In another embodiment, the nucleic acid encoding the gRNA and the nucleic acid encoding the HDR-enhancer are present on separate nucleic acid molecules. In another embodiment, the nucleic acid encoding the Cas9 polypeptide and the nucleic acid encoding the HDR-enhancer are present on a single nucleic acid molecule. In another embodiment, the nucleic acid encoding the Cas9 polypeptide and the nucleic acid encoding the HDR-enhancer are present on separate nucleic acid molecules. In another embodiment, the nucleic acid encoding the gRNA, the nucleic acid encoding the Cas9 polypeptide, and the nucleic acid encoding the HDR-enhancer are present on a single nucleic acid molecule. In another embodiment, the nucleic acid encoding the gRNA and the nucleic acid encoding the Cas9 polypeptide are present on a single nucleic acid molecule and the nucleic acid encoding the HDR-enhancer is present on a separate nucleic acid molecule. In another embodiment, the nucleic acid encoding the gRNA and the nucleic acid encoding the HDR-enhancer are present on a single nucleic acid molecule and the nucleic acid encoding the Cas9 polypeptide is present on a separate nucleic acid molecule. In another embodiment, the nucleic acid encoding the Cas9 polypeptide and the nucleic acid encoding the HDR-enhancer are present on a single nucleic acid molecule and the nucleic acid encoding the gRNA is present on a separate nucleic acid molecule. In another embodiment, the nucleic acid encoding the gRNA, the nucleic acid encoding the Cas9 polypeptide, and the nucleic acid encoding the HDR-enhancer are each present on separate nucleic acid molecules. In one embodiment, the single nucleic acid molecule is a circular double stranded DNA molecule. In another embodiment, the single nucleic acid molecule is a linear double stranded DNA molecule.

In one embodiment, one or a plurality of components are provided as a single admixture. In another embodiment, one or a plurality of components are each provided separately from one another. In another embodiment, one or a plurality of components are each provided in separate solutions.

In one embodiment, the HDR-enhancer molecule is an HDR-enhancing gRNA, and the Cas9 molecule is an enzymatically inactive Cas9 molecule (eiCas9).

In one embodiment, the HDR-enhancer molecule is an HDR-enhancing gRNA, and the Cas9 molecule is fused to a transcription activator or a transcription repressor. In one embodiment, the Cas9 molecule is an enzymatically inactive Cas9 molecule (eiCas9). In another embodiment, the Cas9 molecule is an enzymatically active Cas9 molecule (eaCas9).

In one embodiment, the HDR-enhancing gRNA targets the Cas9 molecule to a gene selected from the group consisting of TP53BP1, RIF1, PAXIP1, XRCC6, XRCC5, PRKDC, LIG4, XRCC4, NHEJ1, DCLRElC, BRCA2, RAD51, XRCC1, LIG1, LIG3, POLQ, FBXO18, RTEL1, PARPBP, UIMC1, RAD52, ERCC1, ERCC4, PARP1, BRCA1, RBBP8, EX01, DNA2, MRE11A, RAD50, NBN, MSH2, MSH3, MSH6, MlH1, PMS2, EZH2, KDM4A/JMJD2A, and CKD1.

In one embodiment, the transcription activator is GAL4, VP16, VP64, a p65 subdomain (NFkB), a histone lysine methyltransferase (KMT), a histone lysine demethylate (KDM), a histone lysine acetyltransferase (KAT), a DNA demethylase, or a protein docking element. In one embodiment, the KMT is hSET1A, hSET1B, MLL1, MLL2, MLL3, MLL4, MLL5, ASH1, Trx, Trr, Ash1, SYMD2, NSD1, or DOT1. In one embodiment, the KDM is LSD1/BHC110, JHDM2a/b, UTX, or JMJD3. In one embodiment, the KAT is hGCN4, PCAF, dGCN5/PCAF, Gcn5, CBP, p300, dCBP/NEJ, TAF1, TIP60/PLIP, MOZ/MYST3, MORF/MYST4, Mst2, Sas3, CG1894, HBO1/MYST2, CHM, Mst2, HMOF/MYST1, dMOF, Sas2, Mst2, SRC1, ACTR, P160, or CLOCK. In one embodiment, the DNA demethylase is AID, TET1, DME, DML1, DML2, ROS1. In one embodiment, the protein docking element is FKBP/FRB (S. pombe) or Pil1/Abyl (E. coli).

In one embodiment, the transcription repressor is KRAB, Mad mSIN3 interaction domain, the ERF repressor domain, a histone lysine methyltransferase (KMT), a histone lysine demthylase (KDM), a histone lysine deacetylase, a DNA methylase, a boundary element, or a periphery recruitment element. In one embodiment, the KMT is SUV39H1, SUV39H2, G9A, Pr-SET7/8, SUV4-10H1, PR-set7, Suv4-20, Set9, EZH2, RIZ1, LSD1/BHC110, SpLsd1/Swm1/Saf110, Su(var)3-3, JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1, JMJD2D, Rph1, JARID1A/RBP2, JARID1B/PLU-1, JARID1C/SMCX, JARID1D/SMCY, Lid, Jhn2, or Jmj2. In one embodiment, the histone lysine deacetylase is HDAC1, HDAC2, HDAC3, HDAC8, Rpd3, Hos1, Cir6, HDAC4, HDAC5, HDAC7, HDAC9, Hdal, Cir3, SIRT1, SIRT2, Sir2, Hst1, Hst2, Hst3, HSt4, or HDAC11. In one embodiment, the DNA methylase is Dam, Dcm, M. SssI, DNMT1, DNMT3a/DNMT3b, METI, DRM3, ZMET2, CMT1, or CMT2. In one embodiment, the boundary element is CTCF. In one embodiment, the periphery recruitment element is LaminA or Lamin B.

In another aspect, described herein is a vector comprising a Cas9 system, wherein said Cas9 system is a composition comprising a nucleic acid. In one embodiment, the vector is a viral vector. In one embodiment, the vector is an AAV vector. In one embodiment, the vector is IDLV.

In another aspect, described herein is a reaction mixture comprising a Cas9 system, a cell or population of cells described herein, and a solution. In one embodiment, the solution is a cell growth medium.

In another aspect, described herein is a method of altering the structure of a cell comprising contacting the cell with a composition, kit, or Cas9 system described herein, or a vector described herein, under conditions that allow for alteration of the structure of the cell, thereby altering the structure of the cell. In one embodiment, the structure of the cell is altered by altering the sequence of a target nucleic acid in the cell.

In another aspect, described herein is a method of treating a subject by altering the structure of a cell in the subject, comprising contacting the cell with a composition, kit, or Cas9 system described herein, or a vector described herein, under conditions that allow for alteration of the structure of the cell, thereby treating the subject. In one embodiment, the subject has a disorder that is caused by a mutation in the target nucleic acid.

In another aspect, described herein is a method of promoting DNA repair of a break in a target nucleic acid via an HDR pathway, the method comprising contacting a cell comprising the target nucleic acid with a composition, Cas9 system, or vector described herein under conditions that allow for repair of the break in the target nucleic acid in the cell via an HDR pathway.

In another aspect, described herein is a method of promoting DNA repair of a double strand break in a target nucleic acid in a cell by an HDR pathway, the method comprising contacting the cell with a gRNA molecule, a Cas9 molecule, and a second gRNA molecule, wherein the gRNA molecule and the second gRNA molecule are oriented on the target nucleic acid such that protospacer adjacent motifs (PAMs) are facing out, wherein the Cas9 nickase molecule cuts the target nucleic acid, resulting in a first 5′ overhang and a second 5′ overhang, thereby promoting DNA repair of the double strand break in the target nucleic acid in the cell via an HDR pathway. In one embodiment, the method further comprises contacting the cell with a template nucleic acid, wherein the template nucleic acid is a single stranded oligonucleotide. In one embodiment, the method further comprises contacting the cell with a template nucleic acid, wherein the template nucleic acid is an endogenous nucleic acid.

In one embodiment, the altered sequence of the target nucleic acid is a deletion in the target nucleic acid. In one embodiment, a mutant or disease phenotype is converted to a non-mutant or non-disease phenotype. In one embodiment, altering the sequence of the target nucleic acid comprises creating a break in the target nucleic acid. In one embodiment, the break is a single strand break. In one embodiment, the break is a double strand break. In one embodiment, the double strand break is blunt-ended or comprises one or two overhangs.

In one embodiment, altering the sequence of the target nucleic acid comprises resection. In one embodiment, resection occurs at a double strand break. In another embodiment, resection occurs at a single strand break.

In one embodiment, the sequence of the target nucleic acid is altered via HR-mediated repair, SSA-mediated repair, or alt-HR-mediated repair. In one embodiment, the level of HR-mediated repair, SSA-mediated repair, or alt-HR-mediated repair is increased as compared to the level of HR-mediated repair, SSA-mediated repair, or alt-HR-mediated repair that would occur in the absence of the HDR-enhancer or in the absence of the eaCas9 molecule.

In one embodiment, the method comprises contacting the cell with a nucleic acid encoding DNA encoding the gRNA, and allowing the cell to produce the gRNA. In one embodiment, the method comprises contacting the cell with a nucleic acid encoding the Cas9 molecule, and allowing the cell to produce the Cas9 molecule. In one embodiment, the method comprises contacting the cell with a nucleic acid that encodes both the gRNA and the Cas9 molecule, and allowing the cell to produce the gRNA and the Cas9 molecule. In one embodiment, the method comprises contacting the cell with a nucleic acid that encodes the gRNA, the Cas9 molecule, and the template nucleic acid; and allowing the cell to produce the gRNA and the Cas9 molecule. In one embodiment, the method comprises contacting the cell with a nucleic acid that encodes the HDR-enhancer, and allowing the cell to produce the HDR-enhancer.

In one embodiment, the HDR-enhancer molecule is a chromatin modifying agent. In one embodiment, the chromatin modifying agent is a chromatin modifying agent other than CKD1. In one embodiment, the chromatin at the target nucleic acid is altered.

In one embodiment, the method further comprises assaying the chromatin state of the cell. In one embodiment, the chromatin state of the target nucleic acid is assayed. In another embodiment, assaying cell cycle status of the cell comprises determining whether the cell is in G2 phase.

In one embodiment, the HDR-enhancer molecule is a cell cycle arrest agent. In one embodiment, the cell cycle arrest agent is not a Cdk1 inhibitor. In one embodiment, the cell arrests in G2. In one embodiment, the cell reversibly arrests in G2.

In one embodiment, the method only substantially down-regulates one DNA repair pathway, or wherein the cell is contacted with only one HDR-enhancer.

In one embodiment, the method further comprising contacting the cell with a second gRNA, wherein the gRNA is configured to guide the Cas9 molecule to produce a first break, and the second gRNA is configured to guide a second Cas9 molecule to produce a second break. In one embodiment, the first break is a single strand break and the second break is a single strand break, the first break is a single strand break and the second break is a double strand break, the first break is a double strand break and the second break is a single strand break, or the first break is a double strand break and the second break is a double strand break.

In one embodiment, the method further comprises contacting the cell with a third gRNA, wherein the third gRNA is configured to guide a third Cas9 molecule to produce a third break. In one embodiment, the method further comprises contacting the cell with a fourth gRNA, wherein the fourth gRNA is configured to guide a fourth Cas9 molecule to produce a fourth break.

In one embodiment, the method further comprises a step of removing the cell from the subject's body before contacting the cell with the gRNA, the Cas9 molecule, the template nucleic acid, or the HDR-enhancer. In one embodiment, the method further comprises a step of returning the cell to the subject's body after contacting the cell with the gRNA, the Cas9 molecule, the template nucleic acid, or the HDR-enhancer. In one embodiment, the method further comprises a step of placing the cell in a subject's body after contacting the cell with the gRNA, the Cas9 molecule, the template nucleic acid, or the HDR-enhancer.

In one embodiment, the cell is contacted with the HDR-enhancer after being contacted with one or more of the gRNA, the Cas9 molecule, and/or template nucleic acid.

In one embodiment, the cell is contacted with the HDR-enhancer before being contacted with one or more of the gRNA, the Cas9 molecule, and/or the template nucleic acid.

In one embodiment, the cell is contacted with two or more of the gRNA, the Cas9 molecule, the template nucleic acid, and the HDR-enhancer at substantially the same time. In one embodiment, the cell is contacted with the gRNA and the Cas9 molecule at substantially the same time.

In one embodiment, a target position in the target nucleic acid is altered to comprise the sequence of at least a portion of a template nucleic acid. In one embodiment, the target nucleic acid bears a mutation relative to a corresponding wild-type sequence, and wherein a template nucleic acid comprises the corresponding wild-type sequence. In one embodiment, the target nucleic acid is pathogenic DNA, and wherein a template nucleic acid contains a mutation relative to the pathogenic DNA.

In one embodiment, a subject has a disorder that is caused by a mutation in the target nucleic acid. In one embodiment, the disorder is cancer, a genetic disease, an infectious disease, a disorder caused by aberrant mitochondrial DNA (mtDNA), a metabolic disease, a disorder caused by aberrant cell cycle, a disorder caused by aberrant angiogenesis, a disorder caused by aberrant DNA damage repair, or a pain disorder.

In one embodiment, the method further comprises a step of removing the cell from the subject's body before contacting the cell with the composition or the vector, and a step of returning the cell to the subject's body after contacting the cell with the composition or the vector. In one embodiment, the cell is present in the body of a subject. In one embodiment, the cell is not present in the body of a subject. In one embodiment, the cell is present in a tissue culture vessel.

In one embodiment, the cell is in G1 phase at the time the cell is contacted with the composition or the vector. In one embodiment, the cell is in G1 phase at the time the Cas9 molecule cleaves the target position. In one embodiment, the cell is in G1 phase at the time the Cas9 molecule-mediated break is repaired by HDR.

In one embodiment, the cell is in S phase at the time the cell is contacted with the composition or the vector. In one embodiment, the cell is in S phase at the time a Cas9 molecule cleaves a target position. In one embodiment, the cell is in S phase at the time a Cas9 molecule-mediated break is repaired by HDR.

In one embodiment, the cell is in G2 phase at the time the cell is contacted with the composition or the vector. In one embodiment, the cell is in G2 phase at the time a Cas9 molecule cleaves a target position. In one embodiment, the cell is in G2 phase at the time a Cas9 molecule-mediated break is repaired by HDR.

In one aspect, described herein is a method of producing the composition, cell, population of cells, kit, or Cas9 system described herein, the method comprising: providing one or more of the gRNA molecule and the Cas9 molecule; providing the HDR-enhancer molecule; and admixing one or more of the gRNA molecule and the Cas9 molecule with the HDR-enhancer molecule. In one embodiment, the method further comprises providing a template nucleic acid and admixing one or more of the gRNA molecule, the Cas9 molecule, the HDR-enhancer molecule with the template nucleic acid.

An additional way of promoting genome editing involves the mismatch repair (MMR) pathway. Certain forms of genome editing, such as an alt-HR pathway, can produce a mismatch in the genome. In some cases the MMR pathway “corrects” the mismatch back to the original sequence, which is an undesirable outcome. To safeguard the edit in the genome, one can down-regulate the MMR pathway in the edited cell.

In one aspect, described herein is a Cas9 system comprising a down-regulator of MMR and one or more of a gRNA molecule and a Cas9 molecule. In one embodiment, the down-regulator of MMR is an inhibitor of a factor listed in Table VI.15. In one embodiment, the down-regulator of MMR is an siRNA, an antibody, a small molecule, or an HDR-enhancing gRNA. In one embodiment, the antibody is an intrabody. In one embodiment, the siRNA or the antibody is directed against a factor listed in Table VI.15. In one embodiment, the down-regulator of MMR is an agent of Table VI.16. In one embodiment, the Cas9 system further comprises a template nucleic acid. In one embodiment, the down-regulator of MMR increases the likelihood that a mismatched base pair in the target nucleic acid will be resolved to have a sequence corresponding to the sequence of a template nucleic acid rather than the sequence of the target nucleic acid before the mismatch was created. In one embodiment, the Cas9 system comprises a nucleic acid encoding the down-regulator of MMR and one or more nucleic acids encoding a gRNA or a Cas9 polypeptide. In one embodiment, the nucleic acids are present in one or more vectors. In one embodiment, the one or more vectors is one or more an AAV vectors.

In another aspect, described herein is a reaction mixture comprising a cell or population of cells described herein, and a solution. In one embodiment, the solution is a growth medium.

In another aspect, the described herein is a method of altering the structure of a cell comprising contacting the cell with a Cas9 system described herein, resulting in alteration of the structure of the cell. In one embodiment, the altering the structure of the cell comprises altering the sequence of a target nucleic acid of the cell.

In another aspect, the described herein is a method of treating a subject by altering the structure of a cell in said subject, comprising contacting the cell with a composition, kit, or Cas9 system described herein under conditions that allow for alteration of the structure of the cell, thereby treating the subject.

In other circumstances, it is desirable to provide an environment favoring nucleotide insertions and/or deletions at the break site via an error-prone repair (EPR) pathway such as alt-NHEJ. To cause a cell to favor an EPR pathway, one can omit a template nucleic acid and contact the cell with an agent that enhances an EPR pathway. An EPR enhancer can be, e.g., an agent that inhibits another DNA damage repair pathway, with the result that the cell becomes more likely to use an alt-NHEJ pathway rather than the inhibited pathway. Other EPR-enhancers directly stimulate an EPR pathway.

In another aspect, the invention provides a Cas9 system comprising an error-prone repair (EPR)-enhancer and one or more of a gRNA molecule and a Cas9 molecule. In one embodiment, the Cas9 system does not comprise a template nucleic acid. In one embodiment, the Cas9 system further comprises a template nucleic acid. In one embodiment, the Cas9 system comprises a nucleic acid encoding the EPR-enhancer and one or more nucleic acids encoding the gRNA or the Cas9 polypeptide. In one embodiment, the nucleic acids are present in one or more vectors. In one embodiment, the vector is an AAV vector.

In one embodiment, the EPR-enhancer is a down-regulator of HDR, an up-regulator of alt-NHEJ, an inhibitor of BRCA1, an up-regulator of SSA, a down-regulator of C-NHEJ, or an agent that promotes resection. In one embodiment, the down-regulator of HDR is an inhibitor of a protein of Table VI.1(C) or VI.2 or an agent of Table VI.3. In one embodiment, the up-regulator of alt-NHEJ is a protein of Table VI.9 or VI.1(J), or an amino acid comprising at least 60, 70, 80, 90, 95, 98, 99 or 100% homology with, or which differs by no more than 50, 40, 30, 20, 15, 10, 5, 4, 3, 2, or 1, amino acid residues from a protein of Table VI.9 or VI.1(J). In one embodiment, the inhibitor of BRCA1 is an siRNA or antibody directed against BRCA1. In one embodiment, the up-regulator of SSA is an inhibitor of BRCA2 or RAD51. In one embodiment, the inhibitor is an siRNA, an antibody, or an HDR-enhancing gRNA. In one embodiment, the down-regulator of C-NHEJ is an inhibitor of a protein of Table VI.1(B) or VII.7, or an agent of Table VI.8. In one embodiment, the agent that promotes resection is a recombinant pro-resection protein or an inhibitor of an anti-resection protein. In one embodiment, the pro-resection protein is a nuclease. In one embodiment, the anti-resection protein is 53BP1, Rif1, or PTIP. In one embodiment, the agent that promotes resection is an agent of Table VI.8. In one embodiment, the inhibitor of an anti-resection protein is a dominant negative 53BP1 protein.

In one aspect, provided herein is a method of altering the structure of a cell comprising contacting the cell with a composition, kit, or Cas9 system described herein, resulting in alteration of the structure of the cell.

In another aspect, described herein is a method of treating a subject by altering the structure of a cell in said subject, comprising contacting the cell with a composition, kit, or Cas9 system described herein, resulting in alteration of the sequence of the target nucleic acid. In one embodiment, no template nucleic acid is provided. In one embodiment, the structure of the cell is altered by altering the structure of a target nucleic acid, and wherein the structure of the nucleic acid is altered via alt-NHEJ-mediated repair or SSA-mediated repair.

In some embodiments, the HDR-enhancer molecule is an HDR-enhancer of Section 1 of this Summary, entitled “Exemplary HDR-enhancers.” In embodiments, the gRNA is a gRNA of Section 2 of this Summary, entitled “Characteristics of the gRNA.” In embodiments, the Cas9 molecule is a Cas9 molecule of Section 3 of this Summary, entitled “Characteristics of the Cas9 molecule.” In embodiments, the template nucleic acid is a template nucleic acid of Section 4 of this Summary, entitled “Characteristics of the template.” In embodiments, the cell is a cell of Section 5 of this Summary, entitled “Characteristics of the cell.” In embodiments, one or more of Properties (i)-(x) are present and are as described in Section 6 of this Summary, entitled “Properties (i)-(x) and characterization thereof.” In embodiments, a composition comprises a characteristic set out in Section 9 of this Summary, entitled “Characteristics of nucleic acid compositions.” In embodiments, the composition comprises a characteristic set out in Section 10 of this Summary, entitled “Additional characteristics of Cas9 systems.”

1. Exemplary HDR-Enhancers

In some embodiments, the HDR-enhancer molecule is a down-regulator of HR, a down-regulator of canonical NHEJ, a down-regulator of alt-NHEJ, a down-regulator of an antirecombinant factor, a down-regulator of SSA, a down-regulator of SSBR, a down-regulator of MMR, a chromatin modification agent, a cell cycle arrest compound, an agent capable of promoting resection at a double strand break, a down-regulator of SD-MMEJ, or a down-regulator of blunt EJ. In one embodiment, the HDR-enhancer molecule is a down-regulator of anti-HR (e.g., an inhibitor of a protein which inhibits HR or promotes repression of HR). In some embodiments, other HDR pathways, such as alt-HR and/or SSA, are promoted and/or the HDR-enhancer molecule is capable of promoting other HDR pathways, such as alt-HR and/or SSA, e.g., as compared to what would be seen in the absence of the down-regulator of anti-HR. In some embodiments, the HDR-enhancer molecule is an inhibitor of a component of Table VI.4 or Table VI.1(D). In some embodiments, the HDR-enhancer molecule is an antibody, e.g., an intrabody, or an siRNA, directed, e.g., against a component from Table VI.4 or Table VI.1(D). In other embodiments, the HDR-enhancer molecule is an HDR-enhancing gRNA directed against one of the repressors or activators described in Example 13. In some embodiments, the HDR-enhancer molecule is an agent of Table VI.5. In some embodiments, the HDR-enhancer inhibits Fbh1, PARI, Rap80, miR-155, miR-545, miR-107, miR-1255, miR-148, or miR-193. In some embodiments, the HDR-enhancer that inhibits Fbh1 is an siRNA. In some embodiments, the HDR enhancer that inhibits PARI is an siRNA. In some embodiments, the HDR-enhancer that inhibits RAP80 is an siRNA. In some embodiments, the HDR-enhancer that inhibits miR-155, miR-545, miR-107, miR-1255, miR-148, or miR-193 is an anti-miR.

In some embodiments, the HDR-enhancer molecule is a down-regulator of SSA (e.g., an inhibitor of a protein, which protein promotes SSA). In embodiments, other HDR pathways, such as alt-HR and/or alt-HR are promoted, e.g., as compared to what would be seen in the absence of the down-regulator of SSA. In embodiments the HDR-enhancer molecule is an inhibitor of a component of Table VI.1(E) or VI.11. In embodiments, the HDR-enhancer molecule is an antibody, e.g., an intrabody, or an siRNA, directed, e.g., against a component from Table VI.1(E) or VI.11. In other embodiments, the HDR-enhancer molecule is an HDR-enhancing gRNA directed against one of the repressors or activators described in Example 13. In embodiments the HDR-enhancer molecule is an agent of Table VI.12. In embodiments the HDR-enhancer inhibits Rad52 or ERCC1. In embodiments the HDR-enhancer that inhibits Rad52 or ERCC1 is an siRNA.

In embodiments, the HDR-enhancer molecule is a chromatin modification agent (e.g., an agent that inhibits a chromatin modification protein that promotes a DNA repair pathway). In some embodiments, the chromatin modification agent is not an HDAC, e.g., is not HDAC1 or HDAC2. In embodiments HDR is promoted and/or the HDR-enhancer molecule is capable of promoting HDR, e.g., as compared to what would be seen in the absence of the chromatin modification agent. In embodiments the HDR-enhancer molecule is administered in an amount sufficient to alter chromatin at the target nucleic acid. In embodiments the chromatin at the target nucleic acid is altered. In embodiments, the chromatin modification agent is not an HDAC, e.g., is not HDAC1 or HDAC2. In embodiments, the HDR-enhancer molecule is a modulator, e.g., inhibitor, of a component of Table VI.1(I). In embodiments, the HDR-enhancer molecule is an antibody, e.g., an intrabody, or an siRNA, directed, e.g., against a component from Table VI.1(I). In another embodiment, the HDR-enhancer molecule is an HDR-enhancing gRNA as described herein and in Example 13. In embodiments, the HDR-enhancer molecule is an agent of Table VII. In embodiments, the HDR-enhancer inhibits EZH2 or an HDAC. In embodiments, the HDR-enhancer that inhibits EZH2 is EPZ-6438. In embodiments, the HDR-enhancer that inhibits the HDAC is TCA.

In embodiments, the HDR-enhancer molecule is a down-regulator of SSBR (e.g., an inhibitor of a protein, which protein promotes SSBR). In embodiments, HDR is promoted and/or the HDR-enhancer molecule is capable of promoting HDR, e.g., as compared to what would be seen in the absence of the down-regulator of SSBR. In embodiments, the HDR-enhancer molecule is an inhibitor of a component of Table VI.13 or VI.1(F). In embodiments, the HDR-enhancer molecule is an agent of Table VI.14. In embodiments, the HDR-enhancer molecule is an antibody, e.g., an intrabody or an siRNA, directed, e.g., against a component from Table VI.13 or VI.1(F). In another embodiment, the HDR-enhancer molecule is an HDR-enhancing gRNA as described herein or in Example 13. In embodiments, the HDR-enhancer inhibits a PARP or XRCC1. In embodiments, the HDR-enhancer that inhibits a PARP is selected from: AZD2281, KU-0059436, and BMN673. In embodiments, the HDR-enhancer that inhibits XRCC1 is an siRNA.

In embodiments, the HDR-enhancer molecule is an agent capable of promoting resection and/or promotes resection at a single or double strand break. In embodiments, HDR is promoted and/or the HDR-enhancer capable of promoting resection is capable of promoting HDR, e.g., as compared to what would be seen in the absence of the HDR-enhancer that promotes resection. In embodiments, the HDR-enhancer that promotes resection at a single or double strand break is an endonuclease or exonuclease. In embodiments, the HDR-enhancer that promotes resection is an inhibitor of an anti-resection protein, e.g., an anti-resection protein of Table VI.1(A). In embodiments, the HDR-enhancer molecule is an antibody, e.g., an intrabody, or an siRNA, directed, e.g., against a component from Table VI.1(A). In one embodiment, the HDR-enhancer molecule is an HDR-enhancing gRNA as described herein or in Example 13. In embodiments, the HDR-enhancer that is an inhibitor of an anti-resection protein is an inhibitor of 53BP1, Rif-1, or PTIP. In one embodiment, the inhibitor of an anti-resection protein is a dominant negative 53BP1 protein.

In embodiments, the HDR-enhancer molecule is a down-regulator of SDMMEJ. In embodiments, the HDR-enhancer molecule is an inhibitor of Pol Theta. In embodiments, the HDR-enhancer molecule is a down-regulator of EJ.

In embodiments, the HDR-enhancer molecule is an agent that promotes cell cycle arrest in G2. In embodiments, the HDR-enhancer molecule is administered in an amount sufficient to cause the cell to arrest in G2. In embodiments, the cell arrests in G2, e.g., reversibly arrests in G2. In embodiments, the HDR-enhancer molecule is a CDK1 inhibitor. In embodiments, the HDR-enhancer molecule is not a CDK1-inhibitor. In embodiments, the HDR-enhancer molecule is an agent of Table VIII.

In embodiments, the HDR-enhancer molecule is a down-regulator of C-NHEJ. In embodiments, HDR is promoted and/or the down-regulator of C-NHEJ is capable of promoting HDR, e.g., as compared to what would be seen in the absence of the down-regulator of C-NHEJ. In embodiments, the HDR-enhancer molecule is an inhibitor of a component of Table VI.7 or VI.1(B). In embodiments, the HDR-enhancer molecule is an antibody, e.g., an intrabody, or an siRNA, directed, e.g., against a component from Table VI.7 or VI.1(B). In embodiments, the HDR-enhancer molecule is an HDR-enhancing gRNA as described herein or in Example 13. In embodiments, the HDR-enhancer molecule is an agent of Table VI.8. In embodiments, the HDR-enhancer molecule is an inhibitor of DNA Pk or 53BP1. In embodiments, the HDR-enhancer that inhibits DNA Pk is selected from: NU7441, CC115, and NK314. In embodiments, the HDR-enhancer that inhibits 53BP1 is an siRNA targeting 53BP1. In one embodiment, the inhibitor of an anti-resection protein is a dominant negative 53BP1 protein.

In embodiments, the HDR-enhancer molecule is a down-regulator of alt-NHEJ, e.g., SD-MMEJ. In embodiments, HDR is promoted and/or the down-regulator of alt-NHEJ is capable of promoting HDR, e.g., as compared to what would be seen in the absence of the down-regulator of alt-NHEJ. In embodiments, the HDR-enhancer molecule is an inhibitor of a component of Table VI.9 or Table VI.1(J). In embodiments, the HDR-enhancer molecule is an antibody, e.g., an intrabody or an siRNA, directed, e.g., against a component from Table VI.9 or Table VI.1(J). In embodiments, the HDR-enhancer molecule is an agent of Table VI.10. In embodiments, the HDR-enhancer molecule is an HDR-enhancing gRNA as described herein.

In embodiments, the HDR-enhancer molecule is an up-regulator of HDR. In embodiments, the up-regulator of HDR is a protein selected from: MRE11, RAD50, NBS1, BRCA2, and BRCA1, or an amino acid comprising at least 60, 70, 80, 90, 95, 98, 99 or 100% homology with, or which differs by no more than 50, 40, 30, 20, 15, 10, 5, 4, 3, 2, or 1, amino acid residues from a naturally occurring MRE11, RAD50, NBS1, BRCA2, or BRCA1. In embodiments, the up-regulator of HDR is a protein of Table VI.2 or a protein of Table VI.1(C), or an amino acid comprising at least 60, 70, 80, 90, 95, 98, 99 or 100% homology with, or which differs by no more than 50, 40, 30, 20, 15, 10, 5, 4, 3, 2, or 1, amino acid residues from, a sequence of Table VI.2 or Table VI.1(C). In another embodiment, the up-regulator of HDR is a dominant negative CtIP. A dominant negative CtIP promotes resection in G1 phase.

In embodiments, the HDR-enhancer molecule is a down-regulator of one HDR pathway (e.g., an inhibitor of a protein, which protein promotes HDR). In embodiments, alt-HR or SSA is promoted and/or the down-regulator of HDR is capable of promoting alt-HR or SSA, e.g., as compared to what would be seen in the absence of the down-regulator of HDR. In embodiments, the HDR-enhancer molecule is an inhibitor of a component of Table VI.2 or VI.1(C). In embodiments, the HDR-enhancer molecule is an antibody, e.g., an intrabody, or an siRNA, directed, e.g., against a component from Table VI.2 or VI.1(C). In embodiments, the HDR-enhancer molecule is an agent of Table VI.3. In embodiments, the HDR-enhancer inhibits BRCA2, BRCA1, or RAD51. In embodiments, the HDR-enhancer molecule is an antibody, e.g., an intrabody, or an siRNA, directed, e.g., against a BRCA2, BRCA1, or RAD51. In some embodiments, the HDR-enhancer molecule is an HDR-enhancing gRNA as described herein and in Example 13. In embodiments, the HDR-enhancer molecule is an siRNA directed against BRCA2, BRCA1, or RAD51. In embodiments, the HDR-enhancer molecule is selected from: B02, A03, AI-10, RI-1, RI-2, and IBR2.

In embodiments, HDR-enhancer comprises an siRNA. In embodiments, the HDR-enhancer comprises an siRNA directed against an mRNA that encodes a target. In embodiments, the HDR-enhancer comprises a polypeptide, e.g., an antibody, e.g., an intrabody, optionally comprising a nuclear localization sequence.

In embodiments, the Cas9 system comprises an additional one or more HDR-enhancers, e.g., exactly two or exactly three HDR-enhancers. In embodiments, the HDR-enhancer of and the additional HDR-enhancer are capable of (i) up-regulating the same pathway, or (ii) down-regulating the same pathway.

2. Characteristics of the gRNA

In embodiments, the gRNA comprises a targeting domain, first and second complementary domains, and a proximal domain.

In embodiments, the gRNA is chimeric. In embodiments, the gRNA is modular.

In embodiments, the at least one domain of a preselected length is a targeting domain which is 12-30 nucleotides in length. In embodiments, the targeting domain is at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In embodiments, the targeting domain is at most 20, 19, 18, 17, or 16 nucleotides in length. In embodiments, the first complementarity domain is at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In embodiments, the first complementarity domain is at most 12, 11, 10, 9, 8, or 7 nucleotides in length. In embodiments, the linking domain is at least 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 nucleotides in length. In embodiments, the linking domain is at most 4, 3, or 2 nucleotides in length. In embodiments, the second complementarity domain is at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In embodiments, the second complementarity domain is at most 12, 11, 10, 9, 8, 7, 6, or 5 nucleotides in length. In embodiments, the at least one domain of a preselected length is a domain encompassing the proximal domain and the tail domain, which taken together are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides in length. In an embodiment, the 5′ extension domain is, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4 nucleotides in length. In an embodiment, the 5′ extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length.

In embodiments, the Cas9 system further comprises a second gRNA. In embodiments, the gRNA of (a) is configured to guide the Cas9 molecule of (b) to produce a first break, and a second gRNA is configured to guide a second Cas9 molecule to produce a second break. In one embodiment, the gRNA of (a) and the second gRNA are configured to position the first break and the second break: within 55 nucleotides of one another; at least 25 nucleotides apart; or within 25-65 nucleotides of one another.

3. Characteristics of the Cas9 Molecule

In embodiments, the Cas9 molecule is an eaCas9 molecule. In embodiments, the eaCas9 forms a double strand break in the target nucleic acid.

In embodiments, the Cas9 molecule is a protein selected from Table 111.1, e.g., a Cas9 molecule other than a S. pyogenes Cas9 molecule. In some embodiments, the Cas9 molecule is a S. pyogenes Cas9 molecule. In other embodiments, the Cas9 molecule is a S. aureus Cas9 molecule.

In embodiments, the eaCas9 molecule comprises N-terminal RuvC-like domain cleavage activity and HNH-like domain cleavage activity. In embodiments, the eaCas9 molecule forms a single strand break in a target nucleic acid. In embodiments, the eaCas9 molecule comprises HNH-like domain cleavage activity but has no, or no significant, N-terminal RuvC-like domain cleavage activity. In embodiments, the eaCas9 molecule is an HNH-like domain nickase. In embodiments, the eaCas9 molecule comprises a mutation at D10. In embodiments, the eaCas9 molecule comprises N-terminal RuvC-like domain cleavage activity but has no, or no significant, HNH-like domain cleavage activity. In embodiments, the eaCas9 molecule is an N-terminal RuvC-like domain nickase. In embodiments, the eaCas9 molecule comprises a mutation at H840.

In embodiments, the Cas9 molecule comprises a REC₂ deletion, REC1_(CT) deletion, or a REC1_(SUB) deletion, or any combination thereof. In embodiments, the Cas9 molecule comprises an altered PI domain.

In embodiments, the Cas9 molecule is less than about 1300 amino acids in length. In embodiments, the Cas9 molecule is: less than about 1200, 1100, 1000, 900, or 800 amino acids in length; or between about 800-1300, 900-1200, 900-1100, or 900-1000 amino acids in length.

In embodiments, the Cas9 molecule is a protein selected from Table 111.1 In some embodiments, the Cas9 molecule is not a S. pyogenes Cas9, e.g., does not comprise SEQ ID NO: 2. In one embodiment, the Cas9 molecule is an S. aureus Cas9 molecule. In one embodiment, the Cas9 molecule is an S. pyogenes Cas9 molecule.

In embodiments, the Cas9 system further comprises a second Cas9 molecule. In embodiments, the Cas9 molecule of (b) is a nickase and the second Cas9 molecule is a nickase; the Cas9 molecule of (b) can catalyze a double strand break and the second Cas9 molecule is a nickase; the Cas9 molecule of (b) is a nickase and the second Cas9 molecule can catalyze a double strand break; or the Cas9 molecule of (b) can catalyze a double strand break and the second Cas9 molecule can catalyze a double strand break.

In some embodiments, the Cas9 recognizes a PAM site other than NGG, e.g., other than AGG. In some embodiments, the Cas9 recognizes an inverted PAM site, e.g., a Pam site that faces outward.

In embodiments, the Cas9 molecule targeted by the gRNA of (a) has the same structure, e.g., amino acid sequence, as the Cas9 molecule targeted by the second gRNA. In other embodiments, the Cas9 molecule targeted by the gRNA of (a) has a different structure, e.g., amino acid sequence, as the Cas9 molecule targeted by the second gRNA.

4. Characteristics of the Template

In embodiments, the template nucleic acid comprises, or comprises a part of, a circular nucleic acid. In embodiments, the template nucleic acid is a circular nucleic acid, e.g., a plasmid. In embodiments, the template nucleic acid is a linear nucleic acid. In some embodiments, the template nucleic acid is DNA. In some embodiments, the template nucleic acid is RNA. In embodiments, the template nucleic acid comprises a double stranded sequence or a single strand sequence, e.g., a single stranded oligonucleotide. In one embodiment, the template is a single stranded/double-stranded DNA hybrid. In another embodiment, the template is present on a circular plasmid. In one embodiment, the donor template is in an AAV or an IDLV. In yet another embodiment, the template nucleic acid is an endogenous nucleic acid. In embodiments, the template nucleic acid comprises about 150-200 nucleotides of homology with a target nucleic acid. In embodiments, the template nucleic acid is linear and comprises about 150-200 nucleotides of homology with a target nucleic acid. In embodiments, the 150-200 nucleotides of homology correspond to one side of a break in a target nucleic acid. In embodiments, the 150-200 nucleotides of homology correspond to two sides of a break in a target nucleic acid. In embodiments, the template nucleic acid comprises about 500-2000 nucleotides of homology with a target nucleic acid.

In embodiments, the template nucleic acid is circular and comprises about 500-2000 nucleotides of homology with a target nucleic acid. In embodiments, the 500-2000 nucleotides of homology correspond to one side of a break in a target nucleic acid. In embodiments, the 500-2000 nucleotides of homology correspond to two sides of a break in a target nucleic acid. In embodiments, the template nucleic acid has homology to the target nucleic acid on one side of the break. In embodiments, the template nucleic acid has homology to the target nucleic acid on two sides of the break.

In embodiments, the template nucleic acid comprises a human sequence, e.g., a wild-type human sequence. In embodiments, the template nucleic acid comprises a wild-type human sequence corresponding to a mutation at a target nucleic acid. In embodiments, the template nucleic acid lacks repeated elements such as an Alu sequence or a LINE sequence.

In embodiments, the template nucleic acid comprises a modified nucleic acid.

In embodiments, one or both of the 3′ and 5′ homology arms, each independently has a length of: at least 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides; no more than 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides; or between 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides.

In embodiments, a homology arm (e.g., the 5′ homology arm or the 3′ homology arm, or both) has a 5′ end and a 3′ end and: the 5′ end is at least 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides from the target position, the 5′ end is no more than 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides from the target position, the 5′ end between 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides from the target position, the 3′ end is at least 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides from the target position, the 3′ end is no more than 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides from the target position, or the 3′ end between 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides from the target position. In embodiments, the replacement sequence has a length of: at least 1, 2, 3, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 300, 4000, or 5000 nucleotides, no more than 2, 3, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 300, 4000, or 5000 nucleotides, or between 1-3, 1-5, 1-10 10-20, 20-50, 50-100, 100-200, 200-500, 500-1000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides.

In embodiments, the target nucleic acid bears a mutation relative to a corresponding wild-type sequence, and the template nucleic acid contains the corresponding wild-type sequence. In embodiments, the target nucleic acid is pathogenic DNA, and the template nucleic acid contains a mutation relative to the pathogenic DNA.

In embodiments, the replacement sequence occupies no more than 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the template nucleic acid. In embodiments, the replacement sequence is at least 17, 18, 19, 20, 25, 30, 40, 50, or 100 nucleotides. In embodiments, the replacement sequence is 1 or 2 nucleotides. In another embodiment, the replacement sequence is 1, 10, 20, 30, 40, 50, 75, 100, 200, 250, 300, 500, 750, or 1000 nucleotides.

5. Characteristics of the Cell

In embodiments, the disclosure provides a cell comprising a Cas9 system described herein.

In embodiments, the cell is a eukaryotic cell. The cell may be, e.g., a vertebrate, mammalian, rodent, goat, pig, bird, chicken, turkey, cow, horse, sheep, fish, primate, plant, or human cell. In embodiments, the cell is a plant cell. The plant cell may be, e.g., monocot or dicot. In embodiments, cell is a mammalian cell, e.g., a human cell. In embodiments, the cell is a somatic cell, germ cell, or prenatal cell. In embodiments, the cell is a zygotic, blastocyst or embryonic cell, a stem cell, a mitotically competent cell, or a meiotically competent cell. In embodiments, the cell is not part of a human embryo. In embodiments, the cell is a somatic cell.

In embodiments, the cell is situated in a subject's body. In embodiments, the cell is not situated in a subject's body. In embodiments, the cell is situated in a tissue culture vessel.

In embodiments, the cell is a T cell, a Hematopoietic Stem Cell, a retinal cell, a cochlear hair cell, a pulmonary epithelial cell, a muscle cell, a neuron, a mesenchymal stem cell, an induced pluripotent stem (iPS) cell, or an embryonic stem cell. In embodiments, the cell is a T cell, a CD8+ T cell, a CD8+ naïve T cell, a central memory T cell, an effector memory T cell, a CD4+ T cell, a stem cell memory T cell, a helper T cell, a regulatory T cell, a cytotoxic T cell, a natural killer T cell, a Hematopoietic Stem Cell, a long term hematopoietic stem cell, a short term hematopoietic stem cell, a multipotent progenitor cell, a lineage restricted progenitor cell, a lymphoid progenitor cell, a myeloid progenitor cell, a common myeloid progenitor cell, an erythroid progenitor cell, a megakaryocyte erythroid progenitor cell, a retinal cell, a photoreceptor cell, a rod cell, a cone cell, a retinal pigmented epithelium cell, a trabecular meshwork cell, a cochlear hair cell, an outer hair cell, an inner hair cell, a pulmonary epithelial cell, a bronchial epithelial cell, an alveolar epithelial cell, a pulmonary epithelial progenitor cell, a striated muscle cell, a cardiac muscle cell, a muscle satellite cell, a neuron, a neuronal stem cell, a mesenchymal stem cell, an induced pluripotent stem (iPS) cell, an embryonic stem cell, a monocyte, a megakaryocyte, a neutrophil, an eosinophil, a basophil, a mast cell, a reticulocyte, a B cell, e.g., a progenitor B cell, a Pre B cell, a Pro B cell, a memory B cell, a plasma B cell, a gastrointestinal epithelial cell, a biliary epithelial cell, a pancreatic ductal epithelial cell, an intestinal stem cell, a hepatocyte, a liver stellate cell, a Kupffer cell, an osteoblast, an osteoclast, an adipocyte, a preadipocyte, a pancreatic islet cell (e.g., a beta cell, an alpha cell, a delta cell), a pancreatic exocrine cell, a Schwann cell, or an oligodendrocyte.

In embodiments, the cell is in G1 phase: at the time the cell is contacted with one or more of (a), (b), (c), and (d); at the time a Cas9 molecule cleaves a target position, or at the time a Cas9 molecule-mediated break is repaired by HDR. In embodiments, the cell is in S phase: at the time the cell is contacted with one or more of (a), (b), (c), and (d); at the time a Cas9 molecule cleaves a target position, or at the time a Cas9 molecule-mediated break is repaired by HDR.

In embodiments, the cell is in G2 phase: at the time the cell is contacted with one or more of (a), (b), (c), and (d); at the time a Cas9 molecule cleaves a target position, or at the time a Cas9 molecule-mediated break is repaired by HDR.

6. Properties (i)-(xi) and Characterization Thereof

In some embodiments, one or more of the following properties is present:

-   -   i. the gRNA is configured to position a Cas9 molecule-mediated         cleavage event at a preselected position relative to a landmark         on the target nucleic acid, wherein the landmark is a site,         e.g., a preselected site in the target nucleic acid, wherein the         target position or the landmark or both are present on an         endogenous chromosomal segment, e.g., are not part of a         heterologous reporter gene;     -   ii. the Cas9 system further comprises a second gRNA suitable for         targeting a Cas9 molecule to the target nucleic acid;     -   iii. the gRNA comprises at least one domain of a preselected         length, e.g., a length disclosed herein;     -   iv. the Cas9 molecule is a protein selected from Table 111.1,         e.g., a Cas9 molecule other than a S. pyogenes Cas9 molecule, or         a Cas9 molecule, other than an S. pyogenes Cas9 molecule,         comprising at least 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85,         90, 95, 99, or 100% homology with, or which differs by no more         than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 35,         50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300,         350 or 400, amino acid residues from, the amino acid sequence of         a reference sequences, e.g., naturally occurring Cas9 molecule,         e.g., a Cas9 molecule described in Table 111.1 herein;     -   v. the Cas9 system further comprises a second Cas9 molecule;     -   vi. the Cas9 molecule is less than about 1300 amino acids in         length;     -   vii. the Cas9 molecule comprises a heterologous PI domain;     -   viii. the Cas9 molecule comprises a REC2 deletion, REC1_(CT)         deletion, or a REC1_(SUB) deletion, or any combination thereof;     -   ix. the template nucleic acid has a preselected sequence         comprising a 5′ homology arm, a replacement sequence, and a 3′         homology arm, wherein the replacement sequence corresponds to an         endogenous nucleic acid, e.g., a chromosomal or organellar         nucleic acid, e.g., are not part of a heterologous reporter         gene; and     -   x. the HDR-enhancer molecule is an antibody, e.g., an intrabody,         a miRNA, a siRNA, e.g., an siRNA having a modified nucleotide,         or an antiMiR.     -   xi. the HDR-enhancer molecule is an HDR-enhancing gRNA, e.g., a         gRNA molecule designed to down-regulate non-HDR DNA repair         pathways including, but not limited to, alt-NHEJ or c-NHEJ (see         FIG. 1 ), or designed to up-regulate HDR DNA repair pathways         including, but not limited to, SSA, alt-HR, or HR (see FIG. 1 ).

In some embodiments, one or more of properties (i)-(xi) is present. In some embodiments, one or more of properties (i), (ii), (iv), (v), (vi), (vii), (viii), (x) or (xi) is present. In some embodiments, one or more of properties (i), (ii), (v), (vi), (vii), (viii), (x), or (xi) is present.

In embodiments, the landmark is: (a) the target position, (b) the 5′ end of a target position, (c) the 3′ end of a target position, (d) within a target position, (e) a position on the target nucleic acid that corresponds to: the 5′ end of the replacement sequence; the 3′ end of the replacement sequence; the 5′ end of the template nucleic acid; the 3′ end of the template nucleic acid; within the 5′ homology arm; within the 3′ homology arm; or within the replacement sequence, or (f) an intron/exon boundary, e.g., the intron/exon boundary nearest the target position or within 50, 100 or 200 nucleotides of the target position; (g) in an intron, e.g., the intron nearest to the target position, the intron within which the target position lies, the nearest intron upstream of the target position, the nearest intron downstream of the target position, or an intron within 50, 100, 200, or 500 nucleotides of the target position; (h) in an exon, e.g., the exon nearest to the target position, the exon within which the target position lies, the nearest exon upstream of the target position, the nearest exon downstream of the target position, or an exon within 50, 100, 200, or 500 nucleotides of the target position; (i) the 5′ end of a coding region, e.g., the coding region nearest to the target position, the coding region within which the target position lies, the nearest coding region upstream of the target position, the nearest coding region downstream of the target position, or a coding region within 50, 100, 200, or 500 nucleotides of the target position; (j) the 3′ end of a coding region, e.g., the coding region nearest to the target position, the coding region within which the target position lies, the nearest coding region upstream of the target position, the nearest coding region downstream of the target position, or a coding region within 50, 100, 200, or 500 nucleotides of the target position; (k) within a coding region, e.g., the coding region nearest to the target position, the coding region within which the target position lies, the nearest coding region upstream of the target position, the nearest coding region downstream of the target position, or a coding region within 50, 100, 200, or 500 nucleotides of the target position; (1) the 5′ end of a transcribed region, e.g., the transcribed region nearest to the target position, the transcribed region within which the target position lies, the nearest transcribed region upstream of the target position, the nearest transcribed region downstream of the target position, or a transcribed region within 50, 100, 200, or 500 nucleotides of the target position; (m) the 3′ end of a transcribed region, e.g., the transcribed region nearest to the target position, the transcribed region within which the target position lies, the nearest transcribed region upstream of the target position, the nearest transcribed region downstream of the target position, or a transcribed region within 50, 100, 200, or 500 nucleotides of the target position; (n) within a transcribed region, e.g., the transcribed region nearest to the target position, the transcribed region within which the target position lies, the nearest transcribed region upstream of the target position, the nearest transcribed region downstream of the target position, or a transcribed region within 50, 100, 200, or 500 nucleotides of the target position; (o) the 5′ end of a repeated element, e.g., the repeated element nearest to the target position, the repeated element within which the target position lies, the nearest repeated element upstream of the target position, the nearest repeated element downstream of the target position, or a repeated element within 50, 100, 200, or 500 nucleotides of the target position; (p) the 3′ end of a repeated element, e.g., the repeated element nearest to the target position, the repeated element within which the target position lies, the nearest repeated element upstream of the target position, the nearest repeated element downstream of the target position, or a repeated element within 50, 100, 200, or 500 nucleotides of the target position; or (q) within a repeated element, e.g., the repeated element nearest to the target position, the repeated element within which the target position lies, the nearest repeated element upstream of the target position, the nearest repeated element downstream of the target position, or a repeated element within 50, 100, 200, or 500 nucleotides of the target position.

In embodiments, the target position is in a naturally occurring fusion protein, e.g., an oncogenic fusion of two genes, e.g., BCR-ABL, TEL-AML1, AML1-ETO, or TMPRSS2-ERG. In some embodiments, the target position is in a gene, e.g., a naturally occurring gene, e.g., a gene that is wild-type or is carrying a naturally-occurring mutation.

In embodiments, the preselected position is selected from at the landmark, away from the landmark; within 50, 100, 150, or 200 nucleotides of the landmark; at least 10, 20, 30, 40, or 50 nucleotides away from the landmark; and 10 to 200, 20-200, 30-200, 40-200, 50-200, 10-150, 10-100, or 10-50 nucleotides from the landmark.

In embodiments, the landmark is a target position and the preselected position is selected from: at the landmark, away from the landmark; within 50, 100, 150, or 200 nucleotides of the landmark; at least 10, 20, 30, 40, or 50 nucleotides away from the landmark; and 10 to 200, 20-200, 30-200, 40-200, 50-200, 10-150, 10-100, or 10-50 nucleotides from the landmark.

In embodiments, the at least one domain of a preselected length is a targeting domain which is 12-30 nucleotides in length. In some embodiments, the at least one domain of a preselected length is a targeting domain which is at least 21 nucleotides in length, e.g., 21-30 nucleotides in length. In embodiments, the at least one domain of a preselected length is a domain encompassing the proximal domain and the tail domain, which taken together are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides in length.

In embodiments, the 5′ homology arm or 3′ homology arm, each independently has, or both have, a length of: at least 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides; no more than 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides; or between 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides. In some embodiments, a 5′ or 3′ homology arm of a ssDNA template nucleic acid is 60-200 nucleotides. In some embodiments, a 5′ or 3′ homology arm of a dsDNA template nucleic acid is 500-4000 nucleotides. In embodiments, the 5′ homology arm has a 5′ end and a 3′ end and: the 5′ end is at least 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides from the target position, the 5′ end is no more than 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides from the target position, the 5′ end between 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides from the target position, the 3′ end is at least 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides from the target position, the 3′ end is no more than 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides from the target position, or the 3′ end between 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides from the target position. In embodiments, the 3′ homology arm has a 5′ end and a 3′ end and: the 5′ end is at least 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides from the target position, the 5′ end is no more than 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides from the target position, the 5′ end between 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides from the target position, the 3′ end is at least 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides from the target position, the 3′ end is no more than 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides from the target position, or the 3′ end between 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides from the target position. In embodiments, the replacement sequence has a length of: at least 1, 2, 3, 5, 10, 20, 50, 100, or 200 nucleotides, no more than 2, 3, 5, 10, 20, 50, 100, 200, or 500 nucleotides, or between 1-3, 1-5, 1-10 10-20, 20-50, 50-100, 100-200, or 200-500 nucleotides.

7. Further Method Steps

In embodiments, the method comprises contacting the cell with a Cas9 system as described herein. In embodiments, the method comprises contacting the cell with a vector as described herein. In embodiments, altering the structure of a cell comprises altering the structure of a target nucleic acid of the cell. In embodiments, the sequence of the target nucleic acid is altered. In embodiments, a deletion is created in the target nucleic acid. In embodiments, a mutant or disease phenotype is converted to a non-mutant or non-disease phenotype.

In embodiments, altering the structure of the target nucleic acid comprises HDR-mediated repair, such as alt-HR mediated repair, SSA-mediated repair, or HR-mediated repair. In embodiments, the efficiency of HDR is increased over the level seen in the absence of an HDR-enhancer. In embodiments, altering the structure of the target nucleic acid comprises creating a break in the target nucleic acid, e.g., a single or double strand break. In embodiments, the double strand break is blunt-ended or comprises one or two overhangs. In embodiments, altering the structure of the target nucleic acid comprises resection, e.g., at a single or double strand break.

In embodiments, the chromatin at the target nucleic acid is altered. In embodiments, the method further comprises assaying cell chromatin state of the cell, e.g., chromatin state of the target nucleic acid.

In embodiments, the method further comprises assaying cell cycle status of the cell, e.g., determining whether the cell is in G2 phase.

In embodiments, the method further comprises contacting the cell with an additional one or more HDR-enhancers, e.g., contacting the cell with exactly two or exactly three HDR-enhancers. In embodiments, the HDR-enhancer and the additional HDR-enhancer (i) up-regulate the same pathway, or (ii) down-regulate the same pathway.

In embodiments, the method further comprises contacting the cell with a second gRNA. In embodiments, the gRNA guides the Cas9 molecule to produce a first break, and a second gRNA guides a second Cas9 molecule to produce a second break. In embodiments, the first break is a single strand break and the second break is a single strand break, the first break is a single strand break and the second break is a double strand break, the first break is a double strand break and the second break is a single strand break, or the first break is a double strand break and the second break is a double strand break.

In embodiments, the method further comprises contacting the cell with a second Cas9 molecule. In embodiments, the Cas9 molecule is a nickase and the second Cas9 molecule is a nickase; the Cas9 molecule can catalyze a double strand break and the second Cas9 molecule is a nickase; the Cas9 molecule is a nickase and the second Cas9 molecule can catalyze a double strand break; or the Cas9 molecule can catalyze a double strand break and the second Cas9 molecule can catalyze a double strand break.

In embodiments, the gRNA targets the Cas9 molecule to make a first break and a second gRNA targets a second Cas9 molecule to make a second break. In embodiments, the two breaks are positioned: within 55 nucleotides of one another; at least 25 nucleotides apart; or within 25-65 nucleotides of one another. In embodiments, the first break is a single strand break and the second break is a single strand break; the first break is a single strand break and the second break is a double strand break; the first break is a double strand break and the second break is a single strand break; or the first break is a double strand break and the second break is a double strand break. In embodiments, the Cas9 molecule targeted by the gRNA has the same structure, e.g., amino acid sequence, as the Cas9 molecule targeted by the second gRNA. In embodiments, the Cas9 molecule targeted by the gRNA has a different structure, e.g., amino acid sequence, as the Cas9 molecule targeted by the second gRNA. In embodiments, the Cas9 molecule creates a first break in at a first target position and the second Cas9 molecule creates a second break at a second target position.

In embodiments, the method forms a double-stranded break that is blunt-ended. In embodiments, the method forms a double strand break that comprises one or two overhangs.

In embodiments, the method further comprises contacting the cell with a cell cycle arrest agent. In embodiments, the cell cycle arrest agent arrests cells in G2. In embodiments, the cell cycle arrest agent is a Cdk1 inhibitor. In embodiments, the cell cycle arrest agent is not a Cdk1 inhibitor. In embodiments, the cell cycle arrest agent is an agent of Table VIII.

In embodiments, the method further comprises a step of removing the cell from a subject's body before contacting the cell with the gRNA, the Cas9 molecule, the template nucleic acid, and the HDR-enhancer. In embodiments, the method further comprises a step of returning the cell to the subject's body after contacting the cell with the gRNA, the Cas9 molecule, the template nucleic acid, and the HDR-enhancer. In embodiments, the method further comprises a step of placing the cell in a subject's body after contacting the cell with the gRNA, the Cas9 molecule, the template nucleic acid, and the HDR-enhancer.

In embodiments, contacting the cell with the gRNA comprises contacting the cell with DNA comprising the sequence of the gRNA, and allowing the cell to produce gRNA. In embodiments, contacting the cell with the Cas9 molecule comprises contacting the cell with a nucleic acid (e.g., DNA or RNA) encoding the Cas9 molecule, and allowing the cell to produce the Cas9 molecule. In embodiments, contacting the cell with the HDR-enhancer comprises contacting the cell with a nucleic acid (e.g., DNA or RNA) encoding the HDR-enhancer, and allowing the cell to produce the HDR-enhancer. In embodiments, contacting the cell with the Cas9 molecule, the gRNA, the template nucleic acid, and the HDR-enhancer comprises contacting the cell with a recombinant nucleic acid that comprises or encodes two of the Cas9 molecule, the gRNA, the template nucleic acid, and the HDR-enhancer, e.g., encodes the Cas9 molecule and encodes or comprises the gRNA, encodes the Cas9 molecule and comprises the template nucleic acid, encodes the Cas9 molecule and encodes or comprises the HDR-enhancer, encodes or comprises the gRNA and comprises the template nucleic acid, encodes or comprises the gRNA and encodes or comprises the HDR-enhancer, or comprises the template nucleic acid and encodes or comprises the HDR-enhancer, and allowing the cell to produce the two of the Cas9 molecule, the gRNA the template nucleic acid, and the HDR-enhancer. In embodiments, contacting the cell with the Cas9 molecule, the gRNA, the template nucleic acid, and the HDR-enhance comprises contacting the cell with a recombinant nucleic acid that comprises or encodes at least three, e.g., all, of the Cas9 molecule, the gRNA, the template nucleic acid, and the HDR-enhancer, e.g.: encodes the Cas9 molecule, encodes or comprises the gRNA, and comprises the template nucleic acid; encodes the Cas9 molecule, encodes or comprises the gRNA, and encodes or comprises the HDR-enhancer; encodes the Cas9 molecule, comprises the template nucleic acid, and encodes or comprises the HDR-enhancer; encodes or comprises the gRNA, comprises the template nucleic acid, and encodes or comprises the HDR-enhancer, or encodes the Cas9 molecule, encodes or comprises the gRNA, comprises the template nucleic acid, and encodes or comprises the HDR-enhancer, and allowing the cell to produce the gRNA and the Cas9 molecule.

In embodiments, the cell is contacted with the HDR-enhancer after being contacted with one or more of the gRNA, the Cas9 molecule, and the template nucleic acid. In embodiments, the cell is contacted with the HDR-enhancer before being contacted with one or more of the gRNA, the Cas9 molecule, and the template nucleic acid. In embodiments, the cell is contacted with the gRNA, the Cas9 molecule, the template nucleic acid, and the HDR-enhancer at substantially the same time. In embodiments, the cell is contacted with the gRNA and the Cas9 molecule at substantially the same time.

In embodiments, the target position is altered to take the sequence of at least a portion of the template nucleic acid, e.g., the replacement sequence or a portion thereof.

In embodiments, administering the gRNA comprises administering DNA encoding the gRNA; administering the Cas9 molecule comprises administering DNA or RNA encoding the Cas9 molecule; or administering the gRNA and Cas9 molecules comprises administering a recombinant nucleic acid that encodes both the gRNA and the Cas9 molecule, or any combination thereof.

In embodiments, the HDR-enhancer molecule is administered separately from the gRNA or the Cas9 molecule.

In embodiments, the method comprises a step of removing the cell from a subject's body before contacting the cell with the gRNA, the Cas9 molecule, the template nucleic acid, and the HDR-enhancer, and further comprising a step of returning the cell to the subject's body after contacting the cell with the gRNA, the Cas9 molecule, the template nucleic acid, and the HDR-enhancer.

In embodiments, only one DNA repair pathway is substantially down-regulated or only one inhibitor is contacted with the cell. In embodiments, two DNA repair pathways are substantially downregulated when only one inhibitor is contacted with the cell. In embodiments, three DNA repair pathways are substantially downregulated when only one inhibitor is contacted with the cell.

In embodiments, the cell is in G1 phase: at the time the cell is contacted with the Cas9 system at the time a Cas9 molecule cleaves a target position, or at the time a Cas9 molecule-mediated break is repaired by HDR. In embodiments, the cell is in S phase: at the time the cell is contacted with the Cas9 system; at the time a Cas9 molecule cleaves a target position, or at the time a Cas9 molecule-mediated break is repaired by HDR.

8. Characteristics of the Subject

In embodiments, the subject has a disorder that is caused by a target position in a target nucleic acid. In embodiments, the disorder is cancer, a genetic disease, an infectious disease, a disorder caused by aberrant mtDNA, a metabolic disease, a disorder caused by aberrant cell cycle, a disorder caused by aberrant angiogenesis, a disorder caused by aberrant DNA damage repair, or a pain disorder. In one embodiment, the subject is a human subject.

9. Characteristics of Nucleic Acid Compositions

In embodiments, the Cas9 system comprises one or more of: a nucleic acid encoding a gRNA suitable for targeting a Cas9 molecule to a target nucleic acid in a cell; a nucleic acid encoding a Cas9 molecule; and a nucleic acid that encodes the HDR-enhancer.

In embodiments, nucleic acid encoding a gRNA suitable for targeting a Cas9 molecule to a target nucleic acid in a cell is DNA. In embodiments, the nucleic acid encoding a Cas9 molecule is DNA. In embodiments, the nucleic acid that encodes the HDR-enhancer molecule is DNA. In embodiments, the Cas9 system comprises a template nucleic acid, which template nucleic acid is optionally DNA.

In embodiments, the composition further comprises a nucleic acid that comprises or encodes a second gRNA. In embodiments, the composition further comprises a nucleic acid that encodes a second Cas9 molecule. In embodiments, the composition further comprises a nucleic acid that comprises or encodes a second HDR-enhancer. In embodiments, the composition further comprises a nucleic acid that comprises or encodes a third HDR-enhancer.

In embodiments, the HDR-enhancer molecule is a protein. In embodiments, the HDR-enhancer molecule is an RNA. In other embodiments, the HDR-enhancer molecule is an HDR-enhancing gRNA molecule.

In embodiments, each nucleic acid is a DNA. In embodiments, the nucleic acid molecule encoding the gRNA molecule, and the nucleic acid molecule encoding the Cas9 molecule are disposed on a single nucleic acid molecule. In other embodiments, the nucleic acid molecule encoding the gRNA molecule, and the nucleic acid molecule encoding the Cas9 molecule are disposed on separate nucleic acid molecules. In one embodiment, the nucleic acid molecule encoding the gRNA molecule, and the nucleic acid molecule encoding the template nucleic acid are disposed on a single nucleic acid molecule. In one embodiment, the nucleic acid molecule encoding the gRNA molecule, and the nucleic acid molecule encoding the template nucleic acid are disposed on separate nucleic acid molecules. In another embodiment, the nucleic acid encoding the Cas9 molecule and the nucleic acid encoding the template nucleic acid are disposed on a single nucleic acid molecule. In another embodiment, the nucleic acid encoding the Cas9 molecule and the nucleic acid encoding the template nucleic acid are disposed on separate nucleic acid molecules. In another embodiment, the nucleic acid encoding the gRNA molecule, the nucleic acid encoding the Cas9 molecule, and the template nucleic acid are disposed on a single nucleic acid molecule. In another embodiment, the nucleic acid encoding the gRNA molecule and the nucleic acid encoding the Cas9 molecule are disposed on a single nucleic acid molecule and the template nucleic acid is disposed on a separate nucleic acid molecule. In another embodiment, the nucleic acid encoding the gRNA molecule and the template nucleic acid are disposed on a single nucleic acid molecule and the nucleic acid encoding the Cas9 molecule is disposed on a separate nucleic acid molecule. In another embodiment, the nucleic acid encoding the Cas9 molecule and the template nucleic acid are disposed on a single nucleic acid molecule and the nucleic acid encoding the gRNA molecule is disposed on a separate nucleic acid molecule. In yet another embodiment, the nucleic acid encoding the gRNA molecule, the nucleic acid encoding the Cas9 molecule, and the template nucleic acid are each disposed on separate nucleic acid molecules.

In embodiments, each nucleic acid forms part of a single nucleic acid molecule. In embodiments, each nucleic acid forms part of a single circular double stranded DNA. In embodiments, each nucleic acid forms part of a linear double stranded DNA.

In embodiments, the composition is a purified composition.

10. Additional Characteristics

In one embodiment, described herein is a cell comprising a Cas9 system described herein. In one embodiment, described herein is a population of cells, each of which comprise a Cas9 system described herein. In one embodiment, described herein is a kit comprising a Cas9 system described herein. In one embodiment, the kit comprises a composition described herein. In another embodiment, the kit comprises a cell or a population of cells described herein.

In one embodiment, described herein is a composition comprising a Cas9 system. In embodiments, the composition further comprises a pharmaceutically acceptable excipient. In embodiments, the gRNA molecule and the Cas9 molecule form part of a single admixture or are provided separately; the gRNA molecule and the HDR-enhancer molecule form part of a single admixture or are provided separately; the gRNA molecule and the template nucleic acid form part of a single admixture or are provided separately; the Cas9 molecule and the HDR-enhancer molecule form part of a single admixture or are provided separately; the Cas9 molecule and the template nucleic acid form part of a single admixture or are provided separately; the HDR-enhancer molecule and the template nucleic acid form part of a single admixture or are provided separately; the gRNA molecule, the Cas9 molecule, and the HDR-enhancer molecule form part of a single admixture or are provided separately; the gRNA molecule, the Cas9 molecule, and the template nucleic acid form part of a single admixture or are provided separately; the Cas9 molecule, the HDR-enhancer molecule, and the template nucleic acid form part of a single admixture or are provided separately; or the gRNA molecule, the Cas9 molecule, the HDR-enhancer molecule, and the template nucleic acid form part of a single admixture or are provided separately.

In embodiments, the Cas9 system comprises a kit. The kit may further comprise packaging. The kit may further comprise instructions for use to treat a disorder, e.g., a disorder caused by a target position in a target nucleic acid. In embodiments, the gRNA molecule and the Cas9 molecule form part of a single admixture or are provided separately; the gRNA molecule and the HDR-enhancer molecule form part of a single admixture or are provided separately; the gRNA molecule and the template nucleic acid form part of a single admixture or are provided separately; the Cas9 molecule and the HDR-enhancer molecule form part of a single admixture or are provided separately; the Cas9 molecule and the template nucleic acid form part of a single admixture or are provided separately; the HDR-enhancer molecule and the template nucleic acid form part of a single admixture or are provided separately; the gRNA molecule, the Cas9 molecule, and the HDR-enhancer molecule form part of a single admixture or are provided separately; the gRNA molecule, the Cas9 molecule, and the template nucleic acid form part of a single admixture or are provided separately; the Cas9 molecule, the HDR-enhancer molecule, and the template nucleic acid form part of a single admixture or are provided separately; or the gRNA molecule, the Cas9 molecule, the HDR-enhancer molecule, and the template nucleic acid form part of a single admixture or are provided separately.

In embodiments, the Cas9 system further comprises a cell cycle arrest agent. The cell cycle arrest agent may be capable of arresting cells in G2 phase. In embodiments, the cell cycle arrest agent is a Cdk1 inhibitor. In embodiments, the inhibitor is an HDR-enhancing gRNA molecule, a small molecule, an siRNA, or an antibody, e.g., intrabody, directed against Cdk1. In embodiments, the cell cycle arrest agent is not a Cdk1 inhibitor.

In embodiments, one or a plurality of components, e.g., the gRNA molecule and the template nucleic acid, are provided as a single admixture. In embodiments, one or a plurality of components, e.g., the gRNA molecule and the template nucleic acid, are each provided separately from one another, e.g., as different solutions.

The disclosure contemplates all combinations of any one or more of the foregoing aspects and/or embodiments, as well as combinations with any one or more of the embodiments set forth in the detailed description and examples.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Headings, including numeric and alphabetical headings and subheadings, are for organization and presentation and are not intended to be limiting.

Other features and advantages of the invention will be apparent from the detailed description, drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are first briefly described.

FIG. 1 is a model representing the DNA repair pathways activated in response to a double-stranded break (DSB).

FIG. 2 is a cartoon depicting the different Cas9 variants and their positioning using a single gRNA or dual gRNAs.

FIG. 3 is a graph depicting that a Cas9 mutated in the RUVC domain with a PAM in the opposite orientation leaves a 5′ protruding end that is more prone to be engaged in HR (gene conversion) in the absence of a donor template nucleic acid. The data is a representation of at least four independent experiments with a minimum of 350 reads per condition.

FIG. 4 is a graph depicting that a Cas9 mutated in the RUVC domain with a Pam facing in the opposite orientation leaves a 5′ protruding end that is more prone to be engaged in HDR in the presence of a single-stranded oligonucleotide donor template nucleic acid. The data is a representation of at least four independent experiments with a minimum of 350 reads per condition.

FIG. 5 is a graph depicting that 60% of the gene editing events using wild-type Cas9 (black) resolve in small deletions, typically a signature of c-NHEJ.

FIG. 6 is a graph depicting that double strand breaks (DSB) generated by wild-type Cas9 are predominantly repaired by canonical NHEJ (c-NHEJ).

FIGS. 7A and 7B are graphs depicting that the down-regulation of Artemis leads to an increase in gene correction mediated by a single-stranded oligonucleotide donor (ss-ODN). FIG. 7A depicts a western blot showing down-regulation of Artemis. FIG. 7B depicts quantification of the gene conversion using a single stranded oligonucleotide donor, wild-type Cas9, and gRNAs HBB-8 and HBB-15 against the HBB locus.

FIG. 8 is a western blot showing the down-regulation of Rad80 using siRNA.

FIG. 9 is a model depicting that double-stranded breaks generated by the N863A Cas9 mutant are predominantly are paired by Alt-NHEJ.

FIG. 10 is a graph depicting that the down-regulation of Pol Theta leads to an increase in gene conversion and a decrease in insertions.

FIG. 11 is a model depicting that double-stranded breaks generated by the D10A Cas9 mutant are predominantly repaired by HR.

FIGS. 12A and 12B depict that gene conversions and non-gene correction is dependent on HR. Specifically, FIG. 12A is a western blot showing BRAC2 and Rad51 down-regulation. FIG. 12B is a graph depicting the percentage of modification observed in U2OS cells edited at the HBB locus with D10A Cas9 and 2 gRNAs with or without BRCA2 or Rad51. FF is a negative control.

FIGS. 13A and 13B demonstrate that gene correction is dependent on SSA. FIG. 13A is a Western blot showing down-regulation of Rad52 and ERCC1. FIG. 13B is a graph depicting the effect of down-regulation of Rad52 and ERCC1 on gene correction at the HBB locus in response to a 5′ protruding double strand break generated with the D10A Cas9 mutant.

FIGS. 14A and 14B depict that gene conversion is dependent on EXO1. The left panel of FIG. 14A is a western blot panel showing down-regulation of EXO1 with siRNA. The right panel of FIG. 14A is a western blot showing the levels of Exo1 in cell lines that have been generated by expression of the gRNA and S.a. FIG. 14B shows two graphs depicting the effect of the down-regulation of Exo1 on gene conversion in response to a 5′ protruding double-stranded break generated with the D10A Cas9 mutant.

FIG. 15 is a model depicting the inhibition of chromatin modification.

DEFINITIONS

“Altered PI domain”, as that term is used herein, refers to a PAM-interacting (PI) domain other than the native or endogenous PI domain associated with the naturally occurring Cas9 molecule. For example, a Cas9 molecule comprises an altered PI domain if its PI domain is other than the PI domain naturally associated with the Cas9 core domain of the Cas9 molecule, or if its PI domain is not a naturally occurring PI domain associated with any Cas9 molecule. (Derived, as used in this sense, is not limited to physical derivation or even derivation from a specific source, and does not require a process limitation, but in an embodiment, includes mere structural similarity). An altered PI domain may have less than 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 70, 60, 50, 30, 40, 30, 20, or 10% homology with the native or endogenous PI domain of a subject naturally occurring Cas9 molecule from which the Cas9 core domain is derived. An altered PI domain may have a different RKR motif (the PAM recognition sequence) than that of the native or endogenous PI domain of the Cas9 species that supplies the Cas9 core domain. The RKR motif of an altered PI domain may differ from the RKR motif of the native or endogenous PI domain of the Cas9 core domain by 1, 2, or 3 residues. The RKR motif of the altered PI differs at the first position, the second position, the third position, the first and second positions, the first and third positions, the second and third positions, or all three positions, from the RKR motif of the PI endogenous to or naturally associated with the Cas9 core domain. In an embodiment, an altered PI domain is one having greater homology with the PI domain of a reference or donor naturally occurring Cas9 molecule (a heterologous Cas9) that with the native PI domain of a subject Cas9.

“ALT-HR” or “alternative HR”, or alternative homology repair pathway, as used herein, refers to the process of repairing DNA damage using a homologous nucleic acid (e.g., a sister chromatid or an exogenous nucleic acid, e.g., a template nucleic acid). ALT-HR is distinct from HR in that the process utilizes different pathways from canonical HR, and can be inhibited by the HR mediators, RAD51 and BRCA2. Also, ALT-HR uses a single-stranded or nicked homologous nucleic acid for repair of the break.

“ALT-NHEJ” or “alternative NHEJ”, or alternative non-homologous end joining, as used herein, is a type of alternative end joining repair process, and utilizes a different pathway than that of canonical NHEJ. In alternative NHEJ, a small degree of resection occurs at the break ends on both sides of the break to reveal single-stranded overhangs. Ligation or annealing of the overhangs results in the deletion of sequence. ALT-NHEJ is a category that includes microhomology-mediated end joining (MMEJ), blunt end joining (EJ), and SD-MMEJ (see FIG. 1 ). In MMEJ, microhomologies, or short spans of homologous sequences, e.g., 5 nucleotides or more, on the single-strand are aligned to guide repair, and leads to the deletion of sequence between the microhomologies.

“Amino acids” as used herein encompasses the canonical amino acids as well as analogs thereof.

“Amino acid residues that flank a deletion”, as that phrase is used herein, refers to the amino acid residue that immediately precedes the deletion and the amino acid residue that immediately follows the deletion. By way of example, in a sequence _(CT)1-_(CT)2-_(CT)3-_(CT)7-_(CT)8-_(CT)9, wherein _(CT)4-_(CT)5-_(CT)6 is deleted, the flanking amino acid residues are, _(CT)3 and _(CT)7.

As used herein, an agent that promotes cell cycle “arrest” refers to an agent that causes a cell to cease dividing and to remain in a characteristic phase of the cell cycle. For instance, the agent may cause the cell to arrest in G1 or G2. In embodiments, the agent produces a reversible cell cycle arrest, such that the cell resumes dividing once the agent is withdrawn.

“Canonical NHEJ”, or canonical non-homologous end joining, as used herein, refers to the process of repairing double strand breaks in which the break ends are directly ligated. This process does not require a homologous nucleic acid to guide the repair, and can result in deletion or insertion of one or more nucleotides. This process requires the Ku heterodimer (Ku70/Ku80), the catalytic subunit of DNA-PK (DN-PKcs), and/or DNA ligase XRCC4/LIG4.

“Cas9 molecule,” as that term is used herein, refers to a Cas9 polypeptide or a nucleic acid encoding a Cas9 polypeptide. A Cas9 polypeptide is a polypeptide that can bind (1) a PAM (a protospacer adjacent motif) in a nucleic acid and (2) a guide RNA (gRNA) molecule. In an embodiment, in concert with the gRNA molecule, a Cas9 polypeptide can localize to a site which comprises a target domain.

A Cas9 molecule may be a nuclease (an enzyme that cleaves both strands of a double-stranded nucleic acid), a nickase (an enzyme that cleaves one strand of a double-stranded nucleic acid), or an enzymatically inactive (or dead) Cas9 molecule. A Cas9 molecule having nuclease or nickase activity is referred to as an “enzymatically active Cas9 molecule” (an “eaCas9” molecule). A Cas9 molecule lacking the ability to cleave target nucleic acid is referred to as an “enzymatically inactive Cas9 molecule” (an “eiCas9” molecule). A Cas9 molecule can have the amino acid sequence of a naturally occurring Cas9 molecule or can be an altered, engineered or modified Cas9 molecule, which differs by at least one amino acid residue, from a reference sequence, e.g., the most similar naturally occurring Cas9 molecule, e.g., a Cas9 molecule from Table 111.1. (The terms altered, engineered or modified, as used in this context, refer merely to a difference from a reference or naturally occurring sequence, and impose no specific process or origin limitations.) A Cas9 molecule may be a Cas9 polypeptide or a nucleic acid encoding a Cas9 polypeptide.

In an embodiment, a Cas9 molecule meets one or both of the following criteria: it has at least 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology with, or it differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 35, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350 or 400, amino acid residues from, the amino acid sequence of a reference sequences, e.g., naturally occurring Cas9 molecule, e.g., a Cas9 molecule described in Table 111.1 herein.

In one embodiment, the Cas9 molecule may be a Cas9 deletion, e.g., the Cas9 may comprise a deletion in one or more of the following domains: a REC2, REC1_(CT), or REC1SUB domain, and optionally, a linker disposed between the amino acids flanking the deletion. Except for any REC deletion and associated linker, a Cas9 molecule meets one or both of the following criteria: it has at least 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 95, 99, or 100% homology with, or it differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 35, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350 or 400, amino acid residues from, the amino acid sequence of a reference sequences, e.g., naturally occurring Cas9 molecule, e.g., a Cas9 molecule described in Table 111.1 herein. Homology except for any REC deletion is determined as follows: a sequence having a deletion is altered by replacing the deleted sequence with the corresponding sequence from the reference sequence, and the altered sequence is compared with the reference sequence.

In another embodiment, the Cas9 molecule may be a Cas9 variant, e.g., the Cas9 molecule may comprise an altered PI domain, or other modified amino acid sequence, or the Cas9 molecule may comprise a linker. In an alternate embodiment, except for an altered PI domain or other modified amino acid sequence, a Cas9 molecule meets one or both of the following criteria: it has at least 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology with, or it differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 35, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350 or 400, amino acid residues from, the amino acid sequence of a reference sequences, e.g., naturally occurring Cas9 molecule, e.g., a Cas9 molecule described in Table 111.1 herein. Homology except for an altered PI domain, or other modified amino acid sequence is determined as follows: a sequence having an altered PI domain (or other modified amino acid sequence) is altered by restoring the altered PI domain (or other modified amino acid sequence) to the naturally occurring PI domain (or other naturally occurring sequence) from the reference sequence, and the thus altered sequence is compared with the reference sequence.

In an alternate embodiment, except for a linker, a Cas9 molecule meets one or both of the following criteria: it has at least 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology with, or it differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 35, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350 or 400, amino acid residues from, the amino acid sequence of a reference sequences, e.g., naturally occurring Cas9 molecule, e.g., a Cas9 molecule described in Table 111.1 herein. Homology except for a linker is determined as follows: a sequence having a linker is altered by omitting the linker sequence, and the thus altered sequence is compared with the reference sequence.

In another embodiment, each domain of the Cas9 molecule (e.g., the domains named herein), including any remaining portion of a REC2, REC1_(CT), or REC1_(SUB) domain having a deletion or an unaltered portion of a PI domain, will, independently have: at least 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology with such a domain described herein, e.g., in a species of Table III.1. In an embodiment at least 1, 2, 3, 4, 5, of 6 domains will have, independently, at least 50, 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology with a corresponding domain, while any remaining domains will be absent, or have less homology to their corresponding naturally occurring domains.

In one embodiment, the Cas9 molecule is a S. pyogenes Cas9 variant. In certain embodiments, the Cas9 variant is the EQR variant. In certain embodiments, the Cas9 variant is the VRER variant. In certain embodiments, the eiCas9 molecule is a S. pyogenes Cas9 variant. In certain embodiments, the Cas9 variant is the EQR variant. In certain embodiments, the Cas9 variant is the VRER variant. In certain embodiments, a Cas9 system comprises a Cas9 molecule, e.g., a Cas9 molecule described herein, e.g., the Cas9 EQR variant or the Cas9 VRER variant.

In some embodiments, the Cas9 molecule is a S. aureus Cas9 variant. In certain embodiments, the Cas9 variant is the KKH (E782K/N968K/R1015H) variant (see Kleinstiver et al. (2015) NAT. BIOTECHNOL. doi: 10.1038/nbt.3404, the entire contents of which are expressly incorporated herein by reference). In some embodiments, the Cas9 variant is the E782K/K929R/R1015H variant (see Kleinstiver et al. (2015)). In some embodiments, the Cas9 variant is the E782K/K929R/N968K/R1015H variant (see Kleinstiver et al. (2015). In some embodiments the Cas9 variant comprises one or more mutations in one of the following residues: E782, K929, N968, R1015. In some embodiments the Cas9 variant comprises one or more of the following mutations: E782K, K929R, N968K, R1015H and R1015Q (see Kleinstiver et al. (2015)). In certain embodiments, a Cas9 system comprises a Cas9 molecule, e.g., a Cas9 molecule described herein, e.g., the Cas9 KKH variant.

“Cas9 polypeptide”, as that term is used herein, also refers to a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology with a reference Cas9 molecule, e.g., a Cas9 molecule of Table 111.1. A Cas9 polypeptide can be enzymatically active (an eaCas9 polypeptide), or can lack the ability to cleave a target nucleic acid (an eiCas9 polypeptide).

“Cas9 core domain”, as that term is used herein, refers to a polypeptide that does not include a functional PI domain, e.g., a polypeptide not having an endogenous PI domain, e.g., wherein the endogenous PI domain is deleted (deleted, as used in this context, refers merely to a sequence difference or the absence of amino acid residues and implies no process or origin limitation), or generally, a Cas9 molecule lacking a PI domain. In an embodiment, a Cas9 core domain comprises a REC1 domain, a REC2 domain, a BH domain, a RuvC domain, and an HNH domain. A Cas9 core domain, together with an altered PI domain, comprises a functional Cas9 molecule.

In an embodiment, a species X Cas9 core domain has at least 20, 30, 40, 50, 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology with the corresponding sequence of a reference sequence, e.g., a naturally occurring species X Cas9 core domain, e.g., from a Cas9 core domain from Table 111.1. In an embodiment, each of a REC1 domain, a REC2 domain, a BH domain, a RuvC domain, and/or an HNH domain of a species X Cas9 core domain has, independently, at least 20, 30, 40, 50, 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology with the corresponding sequence of a reference sequence, e.g., a naturally occurring species X Cas9 core domain, e.g., from a Cas9 core domain from Table III.1.

As used herein, the term “Cas9 system” refers to a system capable of altering a target nucleic acid by one of many DNA repair pathways. In one embodiment, the Cas9 system described herein promotes repair of the target nucleic acid via an HDR pathway. In one embodiment, a Cas9 system comprises a gRNA molecule and a Cas9 molecule. In another embodiment, a Cas9 system comprises a gRNA molecule, a Cas9 molecule, and an HDR-enhancer molecule. In one embodiment, a Cas9 system further comprises a second gRNA molecule. In one embodiment, the Cas9 molecule is fused to a transcription activator. In another embodiment, the Cas9 molecule is fused to a transcription repressor. In yet another embodiment, a Cas9 system comprises a gRNA molecule, a Cas9 nickase molecule, and a second gRNA molecule. In one embodiment, a Cas9 system further comprises a template nucleic acid.

“Derived from”, as used herein, refers to the source or origin of a molecular entity, e.g., a nucleic acid or protein. The source of a molecular entity may be naturally-occurring, recombinant, unpurified, or a purified molecular entity. For example, a polypeptide that is derived from a second polypeptide comprises an amino acid sequence that is identical or substantially similar, e.g., is more than 50% homologous to, the amino acid sequence of the second protein. The derived molecular entity, e.g., a nucleic acid or protein, can comprise one or more modifications, e.g., one or more amino acid or nucleotide changes.

A disorder “caused by” a mutation, as used herein, refers to a disorder that is made more likely or severe by the presence of the mutation, compared to a subject that does not have the mutation. The mutation need not be the only cause of a disorder, i.e., the disorder can still be caused by the mutation even if other causes, such as environmental factors or lifestyle factors, contribute causally to the disorder. In embodiments, the disorder is caused by the mutation if the mutation is a medically recognized risk factor for developing the disorder, and/or if a study has found that the mutation contributes causally to development of the disorder.

“Domain”, as used herein, is used to describe segments of a protein or nucleic acid. Unless otherwise indicated, a domain is not required to have any specific functional property.

A “down-regulator”, as used herein, refers to an agent that directly decreases the activity of a specified biological pathway. Directly decreasing the activity of the pathway refers to the down-regulator binding to a component of that pathway (e.g., a protein that acts in the pathway or an mRNA encoding that protein) and decreasing the level or activity of that component, e.g., by decreasing the concentration or specific activity of that component. For example, a down-regulator may slow one of the steps of that pathway or decrease the level or activity of a component in that pathway. A down-regulator may be, e.g., an inhibitor of a protein in the pathway, or an siRNA or a gRNA that induces a reduction in the expression of a protein in the pathway. The pathway may be, e.g., a DNA damage repair pathway, for example, HDR. In an embodiment, the decreased level or activity is compared to what would be seen in the absence of the down-regulator.

As used herein, “error-prone” repair refers to a DNA repair process that has a higher tendency to introduce mutations into the site being repaired. For instance, alt-NHEJ and SSA are error-prone pathways; C-NHEJ is also error prone because it sometimes leads to the creation of a small degree of alteration of the site (even though in some instances C-NHEJ results in error-free repair); and HR, alt-HR, and SSA in the case of a single strand oligo donor are not error-prone.

As used herein, an “EPR enhancer” refers to an agent that enhances (e.g., increases the frequency or efficiency of) error-prone repair (EPR). In some embodiments, the EPR-enhancer acts on a target in a DNA damage repair pathway, e.g., alt-NHEJ or SSA. The EPR-enhancer may act on, e.g., inhibit, a protein or nucleic acid (e.g., a miRNA) that stimulates a non-error-prone form of DNA repair. The EPR-enhancer may be, e.g., a small molecule, a macromolecule, a protein, an antibody, a peptide, a nucleic acid, a siRNA, an EPR-enhancing gRNA, a miRNA, or an antiMiR.

As used herein, the term “EPR-enhancing gRNA” refers to a gRNA, which, in combination with a Cas9 molecule (e.g., an eiCas9 molecule), enhances (e.g., increases the frequency or efficiency of) error-prone repair (e.g., alt-NJEH and SSA). In some embodiments, the EPR-enhancing gRNA guides a Cas9-mediated reduction in the transcription of a gene encoding a non-error-prone DNA damage repair pathway protein. In some embodiments, the EPR-enhancing gRNA guides a Cas9-mediated cleavage event in a gene encoding a non-error-prone DNA damage repair pathway protein (e.g., a protein involved in HDR, such as HR, alt-HR, and/or SSA).

As used herein, the term “gRNA molecule” or “gRNA” refers to a guide RNA which is capable of targeting a Cas9 molecule to a target nucleic acid. In one embodiment, the term “gRNA molecule” refers to a gRNA. In another embodiment, the term “gRNA molecule” refers to a nucleic acid encoding a gRNA.

“HDR”, or homology-directed repair, as used herein, refers to the process of repairing DNA damage using a homologous nucleic acid (e.g., a sister chromatid or an exogenous nucleic acid, e.g., a template nucleic acid). HDR typically occurs when there has been significant resection at a double strand break, forming at least one single stranded portion of DNA. HDR is a category that includes, for example, single-strand annealing (SSA), homologous recombination (HR), and a third, not yet fully characterized alternative homologous recombination (alt-HR) DNA repair pathway (see FIG. 1 ). In some embodiments, the term HDR does not encompass canonical NHEJ (C-NHEJ). In some embodiments, the term HDR does not encompass alternative non-homologous end joining (Alt-NHEJ) (e.g., blunt end-joining (blunt EJ), (micro homology mediated end joining (MMEJ), and synthesis dependent microhomology-mediated end joining (SD-MMEJ)).

As used herein, the term “HDR-enhancer molecule” or “HDR enhancer” refers to an agent that enhances (e.g., increases the frequency or efficiency of) HDR (e.g., SSA, HR, or alt-HR). In some embodiments, and HDR-enhancer may act on one HDR pathway component to enhance (e.g., increase the frequency or efficiency of) the other HDR pathways. For example, an HDR-enhancer may down-regulate HR in order to enhance SSA and/or alt-HR. In another embodiment, an HDR-enhancer may down-regulate SSA to enhance HR and/or alt-HR. In yet another embodiment, an HDR-enhancer may downregulate alt-HR to enhance HR and/or SSA. In some embodiments, the HDR-enhancer acts to down-regulate a target in a DNA damage repair pathway, e.g., anti-HR, SSA, SSBR, alt-NHEJ, canonical NHEJ, or SDMMEJ. The HDR-enhancer may act on, e.g., inhibit, a protein or nucleic acid (e.g., a miRNA) that stimulates a non-HDR form of DNA repair. The HDR-enhancer molecule may be, e.g., a small molecule, a macromolecule, a protein, an antibody, e.g., an intrabody, a peptide, a nucleic acid, a siRNA, a HDR-enhancing gRNA, a miRNA, or an antiMiR. Alternatively, an HDR-enhancer molecule may be a nucleic acid encoding a protein, a protein, e.g., a dominant negative protein, an antibody, an HDR-enhancing gRNA, a miRNA, or an antiMiR.

As used herein, the term “HDR-enhancing gRNA molecule” or “HDR-enhancing gRNA” refers to a gRNA, which, in combination with a Cas9 molecule (e.g., an eiCas9 molecule or an eaCas9 molecule), enhances (e.g., increases the frequency or efficiency of) HDR (e.g., SSA, HR, or alt-HR) as compared to what would occur in the absence of the HDR-enhancing gRNA molecule. In some embodiments, the HDR-enhancing gRNA molecule guides a Cas9-mediated reduction in the transcription of a gene encoding a DNA damage repair pathway protein. In some embodiments, the HDR-enhancing gRNA molecule guides a Cas9-mediated cleavage event in a gene encoding a DNA damage repair pathway protein. In some embodiments, the DNA damage repair pathway protein is a protein involved in a non-HDR form of DNA repair. In one embodiment, the HDR-enhancing gRNA molecule is a gRNA. In another embodiment, the HDR-enhancing gRNA molecule is a nucleic acid encoding a gRNA.

The terms “homology” or “identity,” as used interchangeably herein, refer to sequence identity between two amino acid sequences or two nucleic acid sequences, with identity being a more strict comparison. The phrases “percent identity or homology” and “% identity or homology” refer to the percentage of sequence identity found in a comparison of two or more amino acid sequences or nucleic acid sequences. Two or more sequences can be anywhere from 0-100% identical, or any value there between. Identity can be determined by comparing a position in each sequence that can be aligned for purposes of comparison to a reference sequence. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position. A degree of identity of amino acid sequences is a function of the number of identical amino acids at positions shared by the amino acid sequences. A degree of identity between nucleic acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. A degree of homology of amino acid sequences is a function of the number of amino acids at positions shared by the polypeptide sequences.

Calculations of homology or sequence identity between two sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frame shift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences.

The term, “HR” refers to a type of HDR DNA-repair which typically acts occurs when there has been significant resection at the double strand break, forming at least one single stranded portion of DNA. In a normal cell, HR” or “Homologous recombination” typically involves a series of steps such as recognition of the break, stabilization of the break, resection, stabilization of single stranded DNA, formation of a DNA crossover intermediate, resolution of the crossover intermediate, and ligation. The process requires RAD51 and BRCA2, and the homologous nucleic acid is typically double-stranded.

The term “inhibitor” as used herein refers to a molecule that binds a specified biological target, thereby inhibiting the function of that biological target. An inhibitor may be, e.g., a small molecule or a siRNA. The biological target may be, e.g., a protein or an RNA (such as an mRNA or a miRNA). In embodiments, the inhibitor is specific for the biological target, e.g., lacks substantial activity against one or more control biological targets. In embodiments, the inhibitor has substantial activity towards only one biological target, or less than 3 biological targets, or less than 5 biological targets. In embodiments, the inhibitor promotes degradation of the biological target.

“Landmark” or “landmark position”, as used herein, refers to a nucleotide in a target nucleic acid.

“Large molecule”, as used herein, refers to a molecule having a molecular weight of at least 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kDa. Large molecules include proteins, polypeptides, nucleic acids, biologics, and carbohydrates.

“Linker”, as that term is used herein, refers to a sequence comprising at least one amino acid. Typically it is disposed between sequences or domains of a Cas9. In an embodiment, the linker is disposed between the amino acid residues that flank a deletion. In an embodiment, the linker is disposed between the amino acid residues of a Cas9 core domain and an altered PI domain. By way of example, in a sequence _(CT)1-_(CT)2-_(CT)3-_(CT)7-_(CT)8-_(CT)9, wherein _(CT)4-_(CT)5-_(CT)6 is deleted, the linker is located immediately C-terminal to the amino acid residue _(CT)3 and immediately N-terminal to the amino acid residue _(CT)7. Preferably, the linker is selected such that the Cas9 molecule exhibits a tertiary structure or folded conformation similar to that of the corresponding naturally occurring Cas9 molecule, such that some Cas9 activity is retained. Suitable linkers are described herein. In an embodiment, the linker comprises a combination of Gly and Ser residues, e.g., (GS)_(x) (SEQ ID NO: 341) or (GGS)_(x)(SEQ ID NO: 339), where x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In an embodiment, the linker comprises a linker comprising the amino acid sequence (SGSETPGTSESATPES)x, where x is 1, 2, 3, or 4 (SEQ ID NO: 344), referred to herein as XTEN linker or XTEN. Alternative linkers include (GSAGSAAGSGEF)_(x), wherein x is 1, 2, 3 or 4 (SEQ ID NO: 201) and (SIVAQLSRPDPA)_(x), wherein x is 1, 2, 3 or 4 (SEQ ID NO: 202). Linkers also include a combination of linkers described herein or known in the art.

“Modulator”, as used herein, refers to an entity, e.g., a compound, that can alter the activity (e.g., enzymatic activity, transcriptional activity, or translational activity), amount, distribution, or structure of a subject molecule or genetic sequence. In an embodiment, modulation comprises cleavage, e.g., breaking of a covalent or non-covalent bond, or the forming of a covalent or non-covalent bond, e.g., the attachment of a moiety, to the subject molecule. In an embodiment, a modulator alters the three dimensional, secondary, tertiary, or quaternary structure, of a subject molecule. A modulator can increase, decrease, initiate, or eliminate a subject activity.

“PI domain”, as that term is used herein, refers to the region of a Cas9 molecule that interacts with the PAM sequence of a target nucleic acid.

“Prevent,” “preventing” and “prevention,” as used herein, means the prevention of a disease in a subject, e.g., a mammal, e.g., in a human, including (a) avoiding or precluding the disease; (2) affecting the predisposition toward the disease, e.g., preventing at least one symptom of the disease or to delay onset of at least one symptom of the disease.

“REC deletion”, as that term is used herein, refers to a REC2 deletion, a REC1_(CT) deletion, or a REC1_(SUB) deletion.

“n” as used herein in the context of proteins or Cas9 molecules described herein, refers to the number of amino acid residues that are deleted in a REC2, REC1_(CT), or REC1_(SUB) deletion, unless otherwise specified.

Unless indicate otherwise, “NHEJ” as used herein encompasses canonical NHEJ and alt-NHEJ.

“Polypeptide”, as used herein, refers to a polymer of amino acids.

“REC2 deletion”, as that term is used herein, refers to a deletion of at least 10% of the amino acid residues of the REC2 domain.

“REC2 domain”, as that term is used herein, refers to a region, in the N terminal half of a naturally occurring Cas9 molecule that is not needed for cleavage or gRNA-mediated targeting. Its length and boundaries differ between Cas9 molecules from various species. In the case of S. aureus, the REC2 domain is about 41 amino acid residues in length and corresponds, approximately, to residues 126 to 166, of S. aureus Cas9. In the case of S. pyogenes, the REC2 domain is about 139 amino acid residues in length and corresponds, approximately, to residues 176 to 314 of S. pyogenes Cas9. In the case of C. jejuni, the REC2 domain is about 45 amino acid residues in length and corresponds, approximately, to residues 137 to 181 of C. jejuni Cas9. These, and the approximate sizes and boundaries of REC2 domains from other species are provided in Table 111.1.

“REC1_(CT) deletion”, as that term is used herein, refers to a deletion of at least 10% of the amino acid residues of the REC1_(CT) domain.

“REC1_(CT) domain”, as that term is used herein, refers to a region, C terminal of the REC1 domain, of a naturally occurring Cas9 polypeptide that is not needed for cleavage or gRNA-mediated targeting. Its length and boundaries differ between Cas9 proteins from various species. In the case of S. aureus, the REC1_(CT) domain is about 146 amino acid residues in length and corresponds, approximately, to residues 288 to 166, of S. aureus Cas9. In the case of S. pyogenes, the REC1_(CT) domain is about 219 amino acid residues in length and corresponds, approximately, to residues 500 to 718 of S. pyogenes Cas9. In the case of C. jejuni, the REC1_(CT) domain is about 134 amino acid residues in length and corresponds, approximately, to residues 305 to 438 of C. jejuni Cas9. These, and the approximate sizes and boundaries of REC1_(CT) domains from other species are provided in Table 111.1.

“REC1_(SUB) deletion”, as that term is used herein, refers to a deletion of at least 10% of the amino acid residues of the REC1_(SUB) domain.

“REC1_(SUB) domain”, as that term is used herein, refers to a region, located within the REC1_(CT) domain, of a naturally occurring Cas9 polypeptide that is not needed for cleavage or gRNA-mediated targeting. Its length and boundaries differ between Cas9 proteins from various species. In the case of S. aureus, the REC1_(Sub) domain is about 57 amino acid residues in length and corresponds, approximately, to residues 296 to 352, of S. aureus Cas9. In the case of S. pyogenes, the REC1_(Sub) domain is about 82 amino acid residues in length and corresponds, approximately, to residues 511 to 592 of S. pyogenes Cas9. In the case of C. jejuni, the REC1_(Sub) domain is about 45 amino acid residues in length and corresponds, approximately, to residues 316 to 360 of C. jejuni Cas9. These, and the approximate sizes and boundaries of REC1_(Sub) domains from other species are provided in Table 111.1.

“Reference molecule”, e.g., a reference Cas9 molecule or reference gRNA, as used herein, refers to a molecule to which a subject molecule, e.g., a subject Cas9 molecule of subject gRNA molecule, e.g., a modified or candidate Cas9 molecule is compared. For example, a Cas9 molecule can be characterized as having no more than 10% of the nuclease activity of a reference Cas9 molecule. Examples of reference Cas9 molecules include naturally occurring unmodified Cas9 molecules, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S. pyogenes, or S. thermophilus. In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology with the Cas9 molecule to which it is being compared. In an embodiment, the reference Cas9 molecule is a sequence, e.g., a naturally occurring or known sequence, which is the parental form on which a change, e.g., a mutation has been made.

“Replacement”, or “replaced”, as used herein with reference to a modification of a molecule does not require a process limitation but merely indicates that the replacement entity is present.

“Resection”, as used herein, refers to exonuclease-mediated digestion of one strand of a double-stranded DNA molecule, which results in a single-stranded overhang. Resection may occur, e.g., on one or both sides of a double-stranded break. Resection can be measured by, for instance, extracting genomic DNA, digesting it with an enzyme that selectively degrades dsDNA, and performing quantitative PCR using primers spanning the DSB site, e.g., as described in Section IV.

“Small molecule”, as used herein, refers to a compound having a molecular weight less than about 2 kDa, e.g., less than about 2 kDa, less than about 1.5 kDa, less than about 1 kDa, or less than about 0.75 kDa.

“Subject”, as used herein, may mean either a human or non-human animal. The term includes, but is not limited to, mammals (e.g., humans, other primates, pigs, rodents (e.g., mice and rats or hamsters), rabbits, guinea pigs, cows, horses, cats, dogs, sheep, and goats). In an embodiment, the subject is a human. In other embodiments, the subject is poultry. In another embodiment, the subject is a fish.

“SSA” or “Single Strand Anealing”, as used herein, refers to the process where RAD52 as opposed to RAD51 in the HR pathways, binds to the single stranded portion of DNA and promotes annealing of the two single stranded DNA segments at repetitive regions. Once RAD52 binds XFP/ERCC1 removes DNA flaps to make the DNA more suitable for ligation.

A “synthetic Cas9 molecule”, or “Syn-Cas9 molecule”, as that term is used herein, refers to a Cas9 molecule that comprises a Cas9 core domain from one bacterial species and a functional altered PI domain, i.e., a PI domain other than that naturally associated with the Cas9 core domain, e.g., from a different bacterial species. Syn-Cas9 polypeptides are also provided.

As used herein, the term “target nucleic acid” refers to a nucleic acid which is being targeted for alteration by a Cas9 system described herein. In one embodiment, a target nucleic acid comprise one gene. In another embodiment, a target nucleic acid may comprise one or more genes, e.g., two genes, three genes, four genes, or five genes.

“Target position” as used herein, refers to a site on a target nucleic acid (e.g., the chromosome) that is modified by a Cas9 molecule-dependent process. For example, the target position can be modified by a Cas9 molecule-mediated cleavage of the target nucleic acid and template nucleic acid directed modification, e.g., correction, of the target position. In an embodiment, a target position can be a site between two nucleotides, e.g., adjacent nucleotides, on the target nucleic acid into which one or more nucleotides is added. The target position may comprise one or more nucleotides that are altered, e.g., corrected, by a template nucleic acid. In an embodiment, the target position is within a “target sequence” (e.g., the sequence to which the gRNA binds). In an embodiment, a target position is upstream or downstream of a target sequence (e.g., the sequence to which the gRNA binds).

The “targeting domain” of the gRNA is complementary to the “target domain” on the target nucleic acid.

A “target sequence” is the sequence of a target domain.

A “template nucleic acid” as that term is used herein, refers to a nucleic acid sequence which can be used in conjunction with a Cas9 molecule and a gRNA molecule to alter the structure of a target position. In an embodiment, the target nucleic acid is modified to have the some or all of the sequence of the template nucleic acid, typically at or near cleavage site(s). In an embodiment, the template nucleic acid is single stranded. In an alternate embodiment, the template nucleic acid is double stranded. In an embodiment, the template nucleic acid is DNA, e.g., double stranded DNA. In an alternate embodiment, the template nucleic acid is single stranded DNA. In an embodiment, the template nucleic acid is RNA, e.g., double stranded RNA or single stranded RNA. In an embodiment, the template nucleic acid is encoded on the same vector backbone, e.g., AAV genome, plasmid DNA, as the Cas9 and gRNA. In an embodiment, the template nucleic acid is excised from a vector backbone in vivo, e.g., it is flanked by gRNA recognition sequences. In one embodiment, the template DNA is in an ILDV. In another embodiment, the template DNA is an endogenous nucleic acid sequence. In one embodiment, the template nucleic acid is a single stranded oligonucleotide corresponding to a plus strand of a nucleic acid sequence. In another embodiment, the template nucleic acid is a single stranded oligonucleotide corresponding to a minus strand of a nucleic acid sequence.

As used herein, the term “transcription activator” refers to a polypeptide or a nucleic acid encoding a polypeptide that increases the transcription of a gene or a set of genes. A transcription activator may be a DNA-binding protein that binds to an enhancer or a promoter-proximal element. In one embodiment, a transcription activator is fused to, or linked to, a Cas9 molecule of the invention in order to temporarily increase transcription of a gene or genes. In one embodiment, the Cas9 molecule is an eaCas9 molecule.

As used herein, the term “transcription repressor” refers to a polypeptide or a nucleic acid encoding a polypeptide that decreases or inhibits the transcription of a gene or a set of genes. A transcription repressor may be a DNA-binding protein that binds to an enhancer or a promoter-proximal element. In one embodiment, a transcription repressor is fused to, or linked to, a Cas9 molecule of the invention in order to temporarily decrease, or temporarily inhibit transcription of a gene or genes. In one embodiment, the Cas9 molecule is an eaCas9 molecule.

“Treat”, “treating” and “treatment”, as used herein, mean the treatment of a disease in a mammal, e.g., in a human, including (a) inhibiting the disease, i.e., arresting or preventing its development; (b) relieving the disease, i.e., causing regression of the disease state; and (c) curing the disease.

An “up-regulator”, as used herein, refers to an agent that directly increases the activity of a specified biological pathway. Directly increasing the activity of the pathway refers to (i) the up-regulator binding to a component of that pathway (e.g., a protein that acts in the pathway or an mRNA encoding that protein) and increasing the level or activity of that component, e.g., by increasing the concentration or specific activity of that component, or (ii) the up-regulator is an added amount of a component that is ordinarily present in the pathway at a given level, e.g., an overexpressed protein. An up-regulator may, e.g., speed up one of the steps of that pathway or increase the level or activity of a component in that pathway. An up-regulator may be, e.g., a protein in the pathway, e.g., one may overexpress a protein that is ordinarily in the pathway to increase the overall activity of the pathway. The pathway may be, e.g., a DNA damage repair pathway, for example, HDR. In an embodiment, the increased level or activity is compared to what would be seen in the absence of the up-regulator.

“Wild type”, as used herein, refers to a gene or polypeptide which has the characteristics, e.g., the nucleotide or amino acid sequence, of a gene or polypeptide from a naturally-occurring source. The term “wild type” typically includes the most frequent observation of a particular gene or polypeptide in a population of organisms found in nature.

“X” as used herein in the context of an amino acid sequence of a linker sequence, refers to any number of repeating units unless otherwise specified.

“X” as used herein in the context of a Cas9 molecule or core domain, e.g., “species X Cas9” designates the species from which the Cas9 molecule or core domain is derived from.

I. gRNA Molecules

A gRNA molecule, as that term is used herein, refers to a nucleic acid that promotes the specific targeting or homing of a gRNA molecule/Cas9 molecule complex to a target nucleic acid. Typically, the nucleic acid will incorporate the functions or structure of both crRNA and tracrRNA, e.g., the functions of processed or mature crRNA and of processed or mature tracrRNA. gRNA molecules can be unimolecular (having a single nucleic acid molecule, e.g., which incorporates both crRNA function or structure and the tracrRNA function or structure), sometimes referred to herein as “chimeric” gRNAs, or modular (comprising more than one, and typically two, separate nucleic acid molecules, e.g., where one incorporates the crRNA function or structure and the other incorporates the tracrRNA function or structure). A gRNA molecule comprises a number of domains. The gRNA molecule domains are described in more detail below. Additional details on gRNAs are provided in Section I entitled “gRNA molecules” of PCT Application WO 2015/048577, the entire contents of which are expressly incorporated herein by reference.

In an embodiment, a unimolecular, or chimeric, gRNA comprises, preferably from 5′ to 3′: a targeting domain (which is complementary to a target nucleic acid, and which is sometimes referred to as a spacer); a first complementarity domain; a linking domain; a second complementarity domain (which is complementary to the first complementarity domain); a proximal domain; and optionally, a tail domain. In an embodiment, the targeting domain, and first complementarity domain correspond functionally or structurally to elements of a crRNA, e.g., a mature or processed crRNA. In an embodiment, the second complementarity domain, proximal domain, and tail domain correspond functionally or structurally to elements of a tracrRNA, e.g., a processed or mature tracrRNA.

In an embodiment, a modular gRNA comprises: a first strand (which corresponds to a crRNA) comprising, preferably from 5′ to 3′; a targeting domain (which is complementary to a target nucleic acid); and a first complementarity domain; and a second strand (which corresponds to a tracrRNA), comprising, preferably from 5′ to 3′: optionally, a 5′ extension domain; a second complementarity domain; a proximal domain; and optionally, a tail domain.

The domains are discussed briefly below.

Targeting Domain

The targeting domain (which can also be referred to as a “spacer”) comprises a nucleotide sequence that is complementary, e.g., at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% complementary, e.g., fully complementary, to the target sequence on the target nucleic acid. The targeting domain is part of an RNA molecule and will therefore comprise the base uracil (U), while any DNA encoding the gRNA molecule will comprise the base thymine (T). While not wishing to be bound by theory, in an embodiment, it is believed that the complementarity of the targeting domain with the target sequence contributes to specificity of the interaction of the gRNA molecule/Cas9 molecule complex with a target nucleic acid. It is understood that in a targeting domain and target sequence pair, the uracil bases in the targeting domain will pair with the adenine bases in the target sequence. In an embodiment, the targeting domain itself comprises in the 5′ to 3′ direction, an optional secondary domain, and a core domain. In an embodiment, the core domain is fully complementary with the target sequence. In an embodiment, the targeting domain is 5 to 50 nucleotides in length, e.g., 10 to 30, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, or 26, nucleotides in length. The strand of the target nucleic acid with which the targeting domain is complementary is referred to herein as the complementary strand. Some or all of the nucleotides of the targeting domain can have a modification, e.g., a modification found in Section X herein.

In an embodiment, the targeting domain is 16 nucleotides in length. In an embodiment, the targeting domain is 17 nucleotides in length. In an embodiment, the targeting domain is 18 nucleotides in length. In an embodiment, the targeting domain is 19 nucleotides in length. In an embodiment, the targeting domain is 20 nucleotides in length. In an embodiment, the targeting domain is 21 nucleotides in length. In an embodiment, the targeting domain is 22 nucleotides in length. In an embodiment, the targeting domain is 23 nucleotides in length. In an embodiment, the targeting domain is 24 nucleotides in length. In an embodiment, the targeting domain is 25 nucleotides in length. In an embodiment, the targeting domain is 26 nucleotides in length. In an embodiment, the targeting domain comprises 16 nucleotides. In an embodiment, the targeting domain comprises 17 nucleotides. In an embodiment, the targeting domain comprises 18 nucleotides. In an embodiment, the targeting domain comprises 19 nucleotides. In an embodiment, the targeting domain comprises 20 nucleotides. In an embodiment, the targeting domain comprises 21 nucleotides. In an embodiment, the targeting domain comprises 22 nucleotides. In an embodiment, the targeting domain comprises 23 nucleotides. In an embodiment, the targeting domain comprises 24 nucleotides. In an embodiment, the targeting domain comprises 25 nucleotides. In an embodiment, the targeting domain comprises 26 nucleotides.

Targeting domains are discussed in more detail below.

First Complementarity Domain

The first complementarity domain is complementary with the second complementarity domain, and in an embodiment, has sufficient complementarity to the second complementarity domain to form a duplexed region under at least some physiological conditions. In an embodiment, the first complementarity domain is 5 to 30 nucleotides in length. In an embodiment, the first complementarity domain is 5 to 25 nucleotides in length. In an embodiment, the first complementary domain is 7 to 25 nucleotides in length. In an embodiment, the first complementary domain is 7 to 22 nucleotides in length. In an embodiment, the first complementary domain is 7 to 18 nucleotides in length. In an embodiment, the first complementary domain is 7 to 15 nucleotides in length. In an embodiment, the first complementary domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.

In an embodiment, the first complementarity domain comprises 3 subdomains, which, in the 5′ to 3′ direction are: a 5′ subdomain, a central subdomain, and a 3′ subdomain. In an embodiment, the 5′ subdomain is 4 to 9, e.g., 4, 5, 6, 7, 8 or 9 nucleotides in length. In an embodiment, the central subdomain is 1, 2, or 3, e.g., 1, nucleotide in length. In an embodiment, the 3′ subdomain is 3 to 25, e.g., 4 to 22, 4 to 18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.

The first complementarity domain can share homology with, or be derived from, a naturally occurring first complementarity domain. In an embodiment, it has at least 50% homology with a first complementarity domain disclosed herein, e.g., an S. pyogenes, S. aureus or S. thermophilus, first complementarity domain.

Some or all of the nucleotides of the domain can have a modification, e.g., a modification found in Section X herein.

First complementarity domains are discussed in more detail below.

Linking Domain

A linking domain serves to link the first complementarity domain with the second complementarity domain of a unimolecular gRNA. The linking domain can link the first and second complementarity domains covalently or non-covalently. In an embodiment, the linkage is covalent. In an embodiment, the linking domain covalently couples the first and second complementarity domains. In an embodiment, the linking domain is, or comprises, a covalent bond interposed between the first complementarity domain and the second complementarity domain. Typically the linking domain comprises one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

In modular gRNA molecules the two molecules are associated by virtue of the hybridization of the complementarity domains.

A wide variety of linking domains are suitable for use in unimolecular gRNA molecules. Linking domains can consist of a covalent bond, or be as short as one or a few nucleotides, e.g., 1, 2, 3, 4, or 5 nucleotides in length. In an embodiment, a linking domain is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in length. In an embodiment, a linking domain is 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, or 2 to 5 nucleotides in length. In an embodiment, a linking domain shares homology with, or is derived from, a naturally occurring sequence, e.g., the sequence of a tracrRNA that is 5′ to the second complementarity domain. In an embodiment, the linking domain has at least 50% homology with a linking domain disclosed herein.

Some or all of the nucleotides of the domain can have a modification, e.g., a modification found in Section X herein.

Linking domains are discussed in more detail below.

5′ Extension Domain

In an embodiment, a modular gRNA can comprise additional sequence, 5′ to the second complementarity domain, referred to herein as the 5′ extension domain. In an embodiment, the 5′ extension domain is, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, or 2 to 4, nucleotides in length. In an embodiment, the 5′ extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length.

Second Complementarity Domain

The second complementarity domain is complementary with the first complementarity domain, and in an embodiment, has sufficient complementarity to the second complementarity domain to form a duplexed region under at least some physiological conditions. In an embodiment, the second complementarity domain can include sequence that lacks complementarity with the first complementarity domain, e.g., sequence that loops out from the duplexed region.

In an embodiment, the second complementarity domain is 5 to 27 nucleotides in length. In an embodiment, it is longer than the first complementarity region. In an embodiment the second complementary domain is 7 to 27 nucleotides in length. In an embodiment, the second complementary domain is 7 to 25 nucleotides in length. In an embodiment, the second complementary domain is 7 to 20 nucleotides in length. In an embodiment, the second complementary domain is 7 to 17 nucleotides in length. In an embodiment, the complementary domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, the second complementarity domain comprises 3 subdomains, which, in the 5′ to 3′ direction are: a 5′ subdomain, a central subdomain, and a 3′ subdomain. In an embodiment, the 5′ subdomain is 3 to 25, e.g., 4 to 22, 4 to 18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In an embodiment, the central subdomain is 1, 2, 3, 4 or 5, e.g., 3, nucleotides in length. In an embodiment, the 3′ subdomain is 4 to 9, e.g., 4, 5, 6, 7, 8 or 9 nucleotides in length.

In an embodiment, the 5′ subdomain and the 3′ subdomain of the first complementarity domain, are respectively, complementary, e.g., fully complementary, with the 3′ subdomain and the 5′ subdomain of the second complementarity domain.

The second complementarity domain can share homology with or be derived from a naturally occurring second complementarity domain. In an embodiment, it has at least 50% homology with a second complementarity domain disclosed herein, e.g., an S. pyogenes, S. aureus or S. thermophilus, first complementarity domain.

Some or all of the nucleotides of the domain can have a modification, e.g., a modification found in Section X herein.

Proximal Domain

In an embodiment, the proximal domain is 5 to 20 nucleotides in length. In an embodiment, the proximal domain can share homology with or be derived from a naturally occurring proximal domain. In an embodiment, it has at least 50% homology with a proximal domain disclosed herein, e.g., an S. pyogenes, S. aureus or S. thermophilus, proximal domain.

Some or all of the nucleotides of the domain can have a modification, e.g., a modification found in Section X herein.

Tail Domain

A broad spectrum of tail domains are suitable for use in gRNA molecules. In an embodiment, the tail domain is 0 (absent), 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In embodiment, the tail domain nucleotides are from or share homology with sequence from the 5′ end of a naturally occurring tail domain. In an embodiment, the tail domain includes sequences that are complementary to each other and which, under at least some physiological conditions, form a duplexed region.

In an embodiment, the tail domain is absent or is 1 to 50 nucleotides in length. In an embodiment, the tail domain can share homology with or be derived from a naturally occurring proximal tail domain. In an embodiment, it has at least 50% homology with a tail domain disclosed herein, e.g., an S. pyogenes, S. aureus or S. thermophilus, tail domain.

In an embodiment, the tail domain includes nucleotides at the 3′ end that are related to the method of in vitro or in vivo transcription. When a T7 promoter is used for in vitro transcription of the gRNA, these nucleotides may be any nucleotides present before the 3′ end of the DNA template. When a U6 promoter is used for in vivo transcription, these nucleotides may be the sequence UUUUUU. When alternate pol-III promoters are used, these nucleotides may be various numbers or uracil bases or may include alternate bases.

In an embodiment the 3′ end of the tail domain is modified to render the gRNA non-toxic to cells or whole organisms e.g., humans.

The domains of gRNA molecules are described in more detail below.

Targeting Domain

The “targeting domain” of the gRNA is complementary to the “target domain” on the target nucleic acid. The strand of the target nucleic acid comprising the nucleotide sequence complementary to the core domain of the gRNA is referred to herein as the “complementary strand” of the target nucleic acid. Guidance on the selection of targeting domains can be found, e.g., in Fu Y et al. (2014) NAT. BIOTECHNOL. 32: 279-84 (doi: 10.1038/nbt.2808) and Sternberg S H et al. (2014) NATURE 507: 62-7 (doi: 10.1038/nature13011).

In an embodiment, the targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, the targeting domain is 16 nucleotides in length. In an embodiment, the targeting domain is 17 nucleotides in length. In an embodiment, the targeting domain is 18 nucleotides in length. In an embodiment, the targeting domain is 19 nucleotides in length. In an embodiment, the targeting domain is 20 nucleotides in length. In an embodiment, the targeting domain is 21 nucleotides in length. In an embodiment, the targeting domain is 22 nucleotides in length. In an embodiment, the targeting domain is 23 nucleotides in length. In an embodiment, the targeting domain is 24 nucleotides in length. In an embodiment, the targeting domain is 25 nucleotides in length. In an embodiment, the targeting domain is 26 nucleotides in length. In an embodiment, the targeting domain comprises 16 nucleotides. In an embodiment, the targeting domain comprises 17 nucleotides. In an embodiment, the targeting domain comprises 18 nucleotides. In an embodiment, the targeting domain comprises 19 nucleotides. In an embodiment, the targeting domain comprises 20 nucleotides. In an embodiment, the targeting domain comprises 21 nucleotides. In an embodiment, the targeting domain comprises 22 nucleotides. In an embodiment, the targeting domain comprises 23 nucleotides. In an embodiment, the targeting domain comprises 24 nucleotides. In an embodiment, the targeting domain comprises 25 nucleotides. In an embodiment, the targeting domain comprises 26 nucleotides.

In an embodiment, the targeting domain is 10+/−5, 20+/−5, 30+/−5, 40+/−5, 50+/−5, 60+/−5, 70+/−5, 80+/−5, 90+/−5, or 100+/−5 nucleotides, in length. In an embodiment, the targeting domain is 20+/−5 nucleotides in length. In an embodiment, the targeting domain is 20+/−10, 30+/−10, 40+/−10, 50+/−10, 60+/−10, 70+/−10, 80+/−10, 90+/−10, or 100+/−10 nucleotides, in length. In an embodiment, the targeting domain is 30+/−10 nucleotides in length. In an embodiment, the targeting domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length. In another embodiment, the targeting domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.

Typically the targeting domain has full complementarity with the target sequence. In an embodiment the targeting domain has or includes 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain.

In an embodiment, the target domain includes 1, 2, 3, 4 or 5 nucleotides that are complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 5′ end. In an embodiment, the target domain includes 1, 2, 3, 4 or 5 nucleotides that are complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 3′ end.

In an embodiment, the target domain includes 1, 2, 3, or 4 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 5′ end. In an embodiment, the target domain includes 1, 2, 3, or 4 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 3′ end.

In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.

In an embodiment, the targeting domain comprises two consecutive nucleotides that are not complementary to the target domain (“non-complementary nucleotides”), e.g., two consecutive noncomplementary nucleotides that are within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or more than 5 nucleotides away from one or both ends of the targeting domain.

In an embodiment, no two consecutive nucleotides within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain, are not complementary to the targeting domain.

In an embodiment, there are no non-complementary nucleotides within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain.

In an embodiment, the targeting domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment, the targeting domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the targeting domain can be modified with a phosphorothioate, or other modification from Section X. In an embodiment, a nucleotide of the targeting domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification(s) from Section X.

In an embodiment, the targeting domain includes 1, 2, 3, 4, 5, 6, 7 or 8 or more modifications. In an embodiment, the targeting domain includes 1, 2, 3, or 4 modifications within 5 nucleotides of its 5′ end. In an embodiment, the targeting domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3′ end.

In an embodiment, the targeting domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or more than 5 nucleotides away from one or both ends of the targeting domain.

In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain.

Modifications in the targeting domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV. gRNAs having a candidate targeting domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in a system in Section IV. The candidate targeting domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In an embodiment, all of the modified nucleotides are complementary to and capable of hybridizing to corresponding nucleotides present in the target domain. In another embodiment, 1, 2, 3, 4, 5, 6, 7 or 8 or more modified nucleotides are not complementary to or capable of hybridizing to corresponding nucleotides present in the target domain.

In an embodiment, the targeting domain comprises, preferably in the 5′→3′ direction: a secondary domain and a core domain. These domains are discussed in more detail below.

Core Domain and Secondary Domain of the Targeting Domain

The “core domain” of the targeting domain is complementary to the “core domain target” on the target nucleic acid. In an embodiment, the core domain comprises about 8 to about 13 nucleotides from the 3′ end of the targeting domain (e.g., the most 3′ 8 to 13 nucleotides of the targeting domain).

In an embodiment, the core domain of the targeting domain and core domain target, are independently, 6+/−2, 7+/−2, 8+/−2, 9+/−2, 10+/−2, 11+/−2, 12+/−2, 13+/−2, 14+/−2, 15+/−2, or 16+−2, nucleotides in length.

In an embodiment, the core domain of the targeting domain and core domain target, are independently, 10+/−2 nucleotides in length.

In an embodiment, the core domain of the targeting domain and core domain target, are independently, 10+/−4 nucleotides in length.

In an embodiment, the core domain of the targeting domain and core domain target are independently 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotides in length.

In an embodiment, the core domain of the targeting domain and core domain target are independently 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20 10 to 20 or 15 to 20 nucleotides in length.

In an embodiment, the core domain of the targeting domain and core domain target are independently 3 to 15, e.g., 6 to 15, 7 to 14, 7 to 13, 6 to 12, 7 to 12, 7 to 11, 7 to 10, 8 to 14, 8 to 13, 8 to 12, 8 to 11, 8 to 10 or 8 to 9 nucleotides in length.

The core domain of the targeting domain is complementary with the core domain target. Typically the core domain has exact complementarity with the core domain target. In an embodiment, the core domain of the targeting domain can have 1, 2, 3, 4 or 5 nucleotides that are not complementary with the corresponding nucleotide of the core domain target. In an embodiment, the degree of complementarity, together with other properties of the gRNA molecule, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.

The “secondary domain” of the targeting domain of the gRNA is complementary to the “secondary domain target” of the target nucleic acid.

In an embodiment, the secondary domain is positioned 5′ to the core domain.

In an embodiment, the secondary domain is absent or optional.

In an embodiment, if the targeting domain is 26 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 13 to 18 nucleotides in length.

In an embodiment, if the targeting domain is 25 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 12 to 17 nucleotides in length.

In an embodiment, if the targeting domain is 24 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 11 to 16 nucleotides in length.

In an embodiment, if the targeting domain is 23 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 10 to 15 nucleotides in length.

In an embodiment, if the targeting domain is 22 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 9 to 14 nucleotides in length.

In an embodiment, if the targeting domain is 21 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 8 to 13 nucleotides in length.

In an embodiment, if the targeting domain is 20 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 7 to 12 nucleotides in length.

In an embodiment, if the targeting domain is 19 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 6 to 11 nucleotides in length.

In an embodiment, if the targeting domain is 18 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 5 to 10 nucleotides in length.

In an embodiment, if the targeting domain is 17 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 4 to 9 nucleotides in length.

In an embodiment, if the targeting domain is 16 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 3 to 8 nucleotides in length.

In an embodiment, the secondary domain is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotides in length.

The secondary domain of the targeting domain is complementary with the secondary domain target. Typically, the secondary domain of the targeting domain has exact complementarity with the secondary domain target. In an embodiment the secondary domain of the targeting domain can have 1, 2, 3, 4 or 5 nucleotides that are not complementary with the corresponding nucleotide of the secondary domain target. In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.

In an embodiment, the core domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment, the core domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the core domain can be modified with a phosphorothioate, or other modification(s) from Section X. In an embodiment a nucleotide of the core domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification(s) from Section X. Typically, a core domain will contain no more than 1, 2, or 3 modifications.

Modifications in the core domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV. gRNAs having a candidate core domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section IV. The candidate core domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In an embodiment, the secondary domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment, the secondary domain comprises one or more modifications, e.g., modifications that render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the secondary domain can be modified with a phosphorothioate, or other modification(s) from Section X. In an embodiment a nucleotide of the secondary domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification from Section X.

Modifications in the secondary domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV. gRNAs having a candidate secondary domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section IV. The candidate secondary domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In an embodiment, (1) the degree of complementarity between the core domain of the targeting domain and its target (i.e., the core domain target), and (2) the degree of complementarity between the secondary domain of the targeting domain and its target (i.e., the secondary domain target), may differ. In an embodiment, (1) may be greater than (2). In an embodiment, (1) may be less than (2). In an embodiment, (1) and (2) are the same, e.g., each may be completely complementary with its target.

In an embodiment, (1) the number of modifications (e.g., modifications from Section X) of the nucleotides of the core domain and (2) the number of modification (e.g., modifications from Section X) of the nucleotides of the secondary domain, may differ. In an embodiment, (1) may be less than (2). In an embodiment, (1) may be greater than (2). In an embodiment, (1) and (2) may be the same, e.g., each may be free of modifications.

First and Second Complementarity Domains

The first complementarity domain is complementary with the second complementarity domain.

Typically the first domain does not have exact complementarity with the second complementarity domain. In an embodiment, the first complementarity domain can have 1, 2, 3, 4 or 5 nucleotides that are not complementary with the corresponding nucleotide of the second complementarity domain. In an embodiment, 1, 2, 3, 4, 5 or 6, e.g., 3 nucleotides, will not pair in the duplex, and, e.g., form a non-duplexed or looped-out region. In an embodiment, an unpaired, or loop-out, region, e.g., a loop-out of 3 nucleotides, is present on the second complementarity domain. In an embodiment, the unpaired region begins 1, 2, 3, 4, 5, or 6, e.g., 4, nucleotides from the 5′ end of the second complementarity domain.

In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.

In an embodiment, the first and second complementarity domains are:

-   -   independently, 6+/−2, 7+/−2, 8+/−2, 9+/−2, 10+/−2, 11+/−2,         12+/−2, 13+/−2, 14+/−2, 15+/−2, 16+/−2, 17+/−2, 18+/−2, 19+/−2,         or 20+/−2, 21+/−2, 22+/−2, 23+/−2, or 24+/−2 nucleotides in         length;     -   independently, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14, 16, 17, 18,         19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length; or     -   independently, 5 to 24, 5 to 23, 5 to 22, 5 to 21, 5 to 20, 7 to         18, 9 to 16, or 10 to 14 nucleotides in length.

In an embodiment, the second complementarity domain is longer than the first complementarity domain, e.g., 2, 3, 4, 5, or 6, e.g., 6, nucleotides longer.

In an embodiment, the first and second complementary domains, independently, do not comprise modifications, e.g., modifications of the type provided in Section X.

In an embodiment, the first and second complementary domains, independently, comprise one or more modifications, e.g., modifications that the render the domain less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the domain can be modified with a phosphorothioate, or other modification(s) from Section X. In an embodiment a nucleotide of the domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification(s) from Section X.

In an embodiment, the first and second complementary domains, independently, include 1, 2, 3, 4, 5, 6, 7 or 8 or more modifications. In an embodiment, the first and second complementary domains, independently, include 1, 2, 3, or 4 modifications within 5 nucleotides of its 5′ end. In an embodiment, the first and second complementary domains, independently, include as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3′ end.

In an embodiment, the first and second complementary domains, independently, include modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5′ end of the domain, within 5 nucleotides of the 3′ end of the domain, or more than 5 nucleotides away from one or both ends of the domain. In an embodiment, the first and second complementary domains, independently, include no two consecutive nucleotides that are modified, within 5 nucleotides of the 5′ end of the domain, within 5 nucleotides of the 3′ end of the domain, or within a region that is more than 5 nucleotides away from one or both ends of the domain. In an embodiment, the first and second complementary domains, independently, include no nucleotide that is modified within 5 nucleotides of the 5′ end of the domain, within 5 nucleotides of the 3′ end of the domain, or within a region that is more than 5 nucleotides away from one or both ends of the domain.

Modifications in a complementarity domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV. gRNAs having a candidate complementarity domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described in Section IV. The candidate complementarity domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In an embodiment, the first complementarity domain has at least 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference first complementarity domain, e.g., a naturally occurring, e.g., an S. pyogenes, S. aureus or S. thermophilus, first complementarity domain, or a first complementarity domain described herein.

In an embodiment, the second complementarity domain has at least 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference second complementarity domain, e.g., a naturally occurring, e.g., an S. pyogenes, S. aureus or S. thermophilus, second complementarity domain, or a second complementarity domain described herein.

The duplexed region formed by first and second complementarity domains is typically 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 base pairs in length (excluding any looped out or unpaired nucleotides).

In an embodiment, the first and second complementarity domains, when duplexed, comprise 11 paired nucleotides, for example, in the gRNA sequence (one paired strand underlined, one bolded):

(SEQ ID NO: 1) NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC.

In an embodiment, the first and second complementarity domains, when duplexed, comprise 15 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):

(SEQ ID NO: 2) NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGAAAAGCAUAGCAA GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCG GUGC.

In an embodiment the first and second complementarity domains, when duplexed, comprise 16 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):

(SEQ ID NO: 3) NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGGAAACAGCAUAGC AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGC.

In an embodiment the first and second complementarity domains, when duplexed, comprise 21 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):

(SEQ ID NO: 4) NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGUUUUGGAAACAAA ACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU GGCACCGAGUCGGUGC.

In an embodiment, nucleotides are exchanged to remove poly-U tracts, for example in the gRNA sequences (exchanged nucleotides underlined):

(SEQ ID NO: 5) NNNNNNNNNNNNNNNNNNNNGUAUUAGAGCUAGAAAUAGCAAGUUAAUAU AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; (SEQ ID NO: 91) NNNNNNNNNNNNNNNNNNNNGUUUAAGAGCUAGAAAUAGCAAGUUUAAAU AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; or (SEQ ID NO: 92) NNNNNNNNNNNNNNNNNNNNGUAUUAGAGCUAUGCUGUAUUGGAAACAAU ACAGCAUAGCAAGUUAAUAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU GGCACCGAGUCGGUGC.

5′ Extension Domain

In an embodiment, a modular gRNA can comprise additional sequence, 5′ to the second complementarity domain. In an embodiment, the 5′ extension domain is 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, or 2 to 4 nucleotides in length. In an embodiment, the 5′ extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length.

In an embodiment, the 5′ extension domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment, the 5′ extension domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the 5′ extension domain can be modified with a phosphorothioate, or other modification(s) from Section X. In an embodiment, a nucleotide of the 5′ extension domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification(s) from Section X.

In an embodiment, the 5′ extension domain can comprise as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications. In an embodiment, the 5′ extension domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 5′ end, e.g., in a modular gRNA molecule. In an embodiment, the 5′ extension domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3′ end, e.g., in a modular gRNA molecule.

In an embodiment, the 5′ extension domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5′ end of the 5′ extension domain, within 5 nucleotides of the 3′ end of the 5′ extension domain, or more than 5 nucleotides away from one or both ends of the 5′ extension domain. In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5′ end of the 5′ extension domain, within 5 nucleotides of the 3′ end of the 5′ extension domain, or within a region that is more than 5 nucleotides away from one or both ends of the 5′ extension domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5′ end of the 5′ extension domain, within 5 nucleotides of the 3′ end of the 5′ extension domain, or within a region that is more than 5 nucleotides away from one or both ends of the 5′ extension domain.

Modifications in the 5′ extension domain can be selected so as to not interfere with gRNA molecule efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV. gRNAs having a candidate 5′ extension domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section IV. The candidate 5′ extension domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In an embodiment, the 5′ extension domain has at least 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference 5′ extension domain, e.g., a naturally occurring, e.g., an S. pyogenes, S. aureus or S. thermophilus, 5′ extension domain, or a 5′ extension domain described herein.

Linking Domain

In a unimolecular gRNA molecule the linking domain is disposed between the first and second complementarity domains. In a modular gRNA molecule, the two molecules are associated with one another by the complementarity domains.

In an embodiment, the linking domain is 10+/−5, 20+/−5, 30+/−5, 40+/−5, 50+/−5, 60+/−5, 70+/−5, 80+/−5, 90+/−5, or 100+/−5 nucleotides, in length.

In an embodiment, the linking domain is 20+/−10, 30+/−10, 40+/−10, 50+/−10, 60+/−10, 70+/−10, 80+/−10, 90+/−10, or 100+/−10 nucleotides, in length.

In an embodiment, the linking domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length.

In another embodiment, the linking domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.

In an embodiment, the linking domain is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 17, 18, 19, or 20 nucleotides in length.

In and embodiment, the linking domain is a covalent bond.

In an embodiment, the linking domain comprises a duplexed region, typically adjacent to or within 1, 2, or 3 nucleotides of the 3′ end of the first complementarity domain and/or the 5-end of the second complementarity domain. In an embodiment, the duplexed region can be 20+/−10 base pairs in length. In an embodiment, the duplexed region can be 10+/−5, 15+/−5, 20+/−5, or 30+/−5 base pairs in length. In an embodiment, the duplexed region can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 base pairs in length.

Typically the sequences forming the duplexed region have exact complementarity with one another, though in an embodiment as many as 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides are not complementary with the corresponding nucleotides.

In an embodiment, the linking domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment, the linking domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the linking domain can be modified with a phosphorothioate, or other modification(s) from Section X. In an embodiment a nucleotide of the linking domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification(s) from Section X. In an embodiment, the linking domain can comprise as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications.

Modifications in a linking domain can be selected so as to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV. gRNAs having a candidate linking domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated a system described in Section IV. A candidate linking domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In an embodiment, the linking domain has at least 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference linking domain, e.g., a linking domain described herein.

Proximal Domain

In an embodiment, the proximal domain is 6+/−2, 7+/−2, 8+/−2, 9+/−2, 10+/−2, 11+/−2, 12+/−2, 13+/−2, 14+/−2, 14+/−2, 16+/−2, 17+/−2, 18+/−2, 19+/−2, or 20+/−2 nucleotides in length.

In an embodiment, the proximal domain is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, the proximal domain is 5 to 20, 7, to 18, 9 to 16, or 10 to 14 nucleotides in length.

In an embodiment, the proximal domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment, the proximal domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the proximal domain can be modified with a phosphorothioate, or other modification(s) from Section X. In an embodiment a nucleotide of the proximal domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification(s) from Section X.

In an embodiment, the proximal domain can comprise as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications. In an embodiment, the proximal domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 5′ end, e.g., in a modular gRNA molecule. In an embodiment, the target domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3′ end, e.g., in a modular gRNA molecule.

In an embodiment, the proximal domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5′ end of the proximal domain, within 5 nucleotides of the 3′ end of the proximal domain, or more than 5 nucleotides away from one or both ends of the proximal domain. In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5′ end of the proximal domain, within 5 nucleotides of the 3′ end of the proximal domain, or within a region that is more than 5 nucleotides away from one or both ends of the proximal domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5′ end of the proximal domain, within 5 nucleotides of the 3′ end of the proximal domain, or within a region that is more than 5 nucleotides away from one or both ends of the proximal domain.

Modifications in the proximal domain can be selected so as to not interfere with gRNA molecule efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV. gRNAs having a candidate proximal domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section IV. The candidate proximal domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In an embodiment, the proximal domain has at least 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference proximal domain, e.g., a naturally occurring, e.g., an S. pyogenes, S. aureus or S. thermophilus, proximal domain, or a proximal domain described herein.

Tail Domain

In an embodiment, the tail domain is 10+/−5, 20+/−5, 30+/−5, 40+/−5, 50+/−5, 60+/−5, 70+/−5, 80+/−5, 90+/−5, or 100+/−5 nucleotides, in length.

In an embodiment, the tail domain is 20+/−5 nucleotides in length.

In an embodiment, the tail domain is 20+/−10, 30+/−10, 40+/−10, 50+/−10, 60+/−10, 70+/−10, 80+/−10, 90+/−10, or 100+/−10 nucleotides, in length.

In an embodiment, the tail domain is 25+/−10 nucleotides in length.

In an embodiment, the tail domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length.

In another embodiment, the tail domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.

In an embodiment, the tail domain is 1 to 20, 1 to 15, 1 to 10, or 1 to 5 nucleotides in length.

In an embodiment, the tail domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment, the tail domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the tail domain can be modified with a phosphorothioate, or other modification(s) from Section X. In an embodiment, a nucleotide of the tail domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification(s) from Section X.

In an embodiment, the tail domain can have as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications. In an embodiment, the target domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 5′ end. In an embodiment, the target domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3′ end.

In an embodiment, the tail domain comprises a tail duplex domain, which can form a tail duplexed region. In an embodiment, the tail duplexed region can be 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 base pairs in length. In an embodiment, a further single stranded domain, exists 3′ to the tail duplexed domain. In an embodiment, this domain is 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In an embodiment it is 4 to 6 nucleotides in length.

In an embodiment, the tail domain has at least 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference tail domain, e.g., a naturally occurring, e.g., an S. pyogenes, S. aureus or S. thermophilus, tail domain, or a tail domain described herein.

In an embodiment, the proximal and tail domain, taken together comprise the following sequences:

(SEQ ID NO: 100) AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU, or (SEQ ID NO: 101) AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGGUGC, or (SEQ ID NO: 102) AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAU C, or (SEQ ID NO: 103) AAGGCUAGUCCGUUAUCAACUUGAAAAAGUG, or (SEQ ID NO: 104) AAGGCUAGUCCGUUAUCA, or (SEQ ID NO: 105) AAGGCUAGUCCG.

In an embodiment, the tail domain comprises the 3′ sequence UUUUUU, e.g., if a U6 promoter is used for transcription.

In an embodiment, the tail domain comprises the 3′ sequence UUUU, e.g., if an H1 promoter is used for transcription.

In an embodiment, tail domain comprises variable numbers of 3′ Us depending, e.g., on the termination signal of the pol-III promoter used.

In an embodiment, the tail domain comprises variable 3′ sequence derived from the DNA template if a T7 promoter is used.

In an embodiment, the tail domain comprises variable 3′ sequence derived from the DNA template, e.g., if in vitro transcription is used to generate the RNA molecule.

In an embodiment, the tail domain comprises variable 3′ sequence derived from the DNA template, e.g., if a pol-II promoter is used to drive transcription.

Modifications in the tail domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV. gRNAs having a candidate tail domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described in Section IV. The candidate tail domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In an embodiment, the tail domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5′ end of the tail domain, within 5 nucleotides of the 3′ end of the tail domain, or more than 5 nucleotides away from one or both ends of the tail domain. In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5′ end of the tail domain, within 5 nucleotides of the 3′ end of the tail domain, or within a region that is more than 5 nucleotides away from one or both ends of the tail domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5′ end of the tail domain, within 5 nucleotides of the 3′ end of the tail domain, or within a region that is more than 5 nucleotides away from one or both ends of the tail domain.

In an embodiment a gRNA has the following structure:

-   -   5′ [targeting domain]-[first complementarity domain]-[linking         domain]-[second complementarity domain]-[proximal domain]-[tail         domain]-3′     -   wherein, the targeting domain comprises a core domain and         optionally a secondary domain, and is 10 to 50 nucleotides in         length;     -   the first complementarity domain is 5 to 25 nucleotides in         length and, In an embodiment has at least 50, 60, 70, 80, 81,         82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,         98, 99% homology with a reference first complementarity domain         disclosed herein;     -   the linking domain is 1 to 5 nucleotides in length;     -   the second complementarity domain is 5 to 27 nucleotides in         length and, in an embodiment has at least 50, 60, 70, 80, 81,         82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,         98, 99% homology with a reference second complementarity domain         disclosed herein;     -   the proximal domain is 5 to 20 nucleotides in length and, in an         embodiment has at least 50, 60, 70, 80, 81, 82, 83, 84, 85, 86,         87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology         with a reference proximal domain disclosed herein; and     -   the tail domain is absent or a nucleotide sequence is 1 to 50         nucleotides in length and, in an embodiment has at least 60, 70,         80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,         96, 97, 98, 99% homology with a reference tail domain disclosed         herein.

Exemplary Chimeric gRNAs

In an embodiment, a unimolecular, or chimeric, gRNA comprises, preferably from 5′ to 3′:

-   -   a targeting domain (which is complementary to a target nucleic         acid);     -   a first complementarity domain, e.g., comprising 15, 16, 17, 18,         19, 20, 21, 22, 23, 24, 25, or 26 nucleotides;     -   a linking domain;     -   a second complementarity domain (which is complementary to the         first complementarity domain);     -   a proximal domain; and     -   a tail domain,     -   wherein,     -   (a) the proximal and tail domain, when taken together, comprise         at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53         nucleotides;     -   (b) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,         50, or 53 nucleotides 3′ to the last nucleotide of the second         complementarity domain; or     -   (c) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,         51, or 54 nucleotides 3′ to the last nucleotide of the second         complementarity domain that is complementary to its         corresponding nucleotide of the first complementarity domain.

In an embodiment, the sequence from (a), (b), or (c), has at least 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology with the corresponding sequence of a naturally occurring gRNA, or with a gRNA described herein.

In an embodiment, the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the unimolecular, or chimeric, gRNA molecule (comprising a targeting domain, a first complementary domain, a linking domain, a second complementary domain, a proximal domain and, optionally, a tail domain) comprises the following sequence in which the targeting domain is depicted as 20 Ns but could be any sequence and range in length from 16 to 26 nucleotides and in which the gRNA sequence is followed by 6 Us, which serve as a termination signal for the U6 promoter, but which could be either absent or fewer in number:

(SEQ ID NO: 106) NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU UU. In an embodiment, the unimolecular, or chimeric, gRNA molecule is a S. pyogenes gRNA molecule.

In some embodiments, the unimolecular, or chimeric, gRNA molecule (comprising a targeting domain, a first complementary domain, a linking domain, a second complementary domain, a proximal domain and, optionally, a tail domain) comprises the following sequence in which the targeting domain is depicted as 20 Ns but could be any sequence and range in length from 16 to 26 nucleotides and in which the gRNA sequence is followed by 6 Us, which serve as a termination signal for the U6 promoter, but which could be either absent or fewer in number:

(SEQ ID NO: 107) NNNNNNNNNNNNNNNNNNNNGUUUUAGUACUCUGGAAACAGAAUCUACUA AAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU UU. In an embodiment, the unimolecular, or chimeric, gRNA molecule is a S. aureus gRNA molecule.

Exemplary Modular gRNAs

In an embodiment, a modular gRNA comprises:

-   -   a first strand comprising, preferably from 5′ to 3′;     -   a targeting domain, e.g., comprising 15, 16, 17, 18, 19, 20, 21,         22, 23, 24, 25, or 26 nucleotides;     -   a first complementarity domain; and     -   a second strand, comprising, preferably from 5′ to 3′:     -   optionally a 5′ extension domain;     -   a second complementarity domain;     -   a proximal domain; and     -   a tail domain,     -   wherein:     -   (a) the proximal and tail domain, when taken together, comprise         at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53         nucleotides;     -   (b) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,         50, or 53 nucleotides 3′ to the last nucleotide of the second         complementarity domain; or     -   (c) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,         51, or 54 nucleotides 3′ to the last nucleotide of the second         complementarity domain that is complementary to its         corresponding nucleotide of the first complementarity domain.

In an embodiment, the sequence from (a), (b), or (c), has at least 60, 75, 80, 85, 90, 95, or 99% homology with the corresponding sequence of a naturally occurring gRNA, or with a gRNA described herein.

In an embodiment, the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 5 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides. In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In another aspect, methods and compositions discussed herein provide methods and compositions for gene editing by using a gRNA molecule which comprises a polyA tail. In one embodiment, a polyA tail of undefined length ranging from 1 to 1000 nucleotide is added enzymatically using a polymerase such as E. coli polyA polymerase (E-PAP). In one embodiment, the polyA tail of a specified length (e.g., 1, 5, 10, 20, 30, 40, 50, 60, 100, or 150 nucleotides (SEQ ID NO: 2289)) is encoded on a DNA template and transcribed with the gRNA via an RNA polymerase (e.g., T7 RNA polymerase). In one embodiment, a polyA tail of defined length (e.g., 1, 5, 10, 20, 30, 40, 50, 60, 100, or 150 nucleotides (SEQ ID NO: 2289)) is synthesized as a synthetic oligonucleotide and ligated on the 3′ end of the gRNA with either an RNA ligase or a DNA ligase with our without a splinted DNA oligonucleotide complementary to the guide RNA and the polyA oligonucleotide. In one embodiment, the entire gRNA including a defined length of polyA tail is made synthetically, in one or several pieces, and ligated together by either an RNA ligase or a DNA ligase with or without a splinted DNA oligonucleotide.

Additional exemplary gRNAs for use in the present invention are disclosed in International Application WO 2015/048577, the entire contents of which are expressly incorporated herein by reference.

In embodiments, one or more of the gRNA domains (e.g., the targeting domain, first complementarity domain, linking domain, second complementarity domain, proximal domain, or tail domain) has at least 50, 60, 70, 80, 85, 90, or 95% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a corresponding reference domain, e.g., a naturally occurring domain of a bacterial strain disclosed herein.

In an embodiment, one or more of the gRNA domains (e.g., the targeting domain, first complementarity domain, linking domain, second complementarity domain, proximal domain, or tail domain), independently, do not comprise modifications. In an embodiment, one or more of the gRNA domains (e.g., the targeting domain, first complementarity domain, linking domain, second complementarity domain, proximal domain, or tail domain), independently, comprise one or more modifications, e.g., modifications that the render the domain less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the domain can be modified with a phosphorothioate. In an embodiment a nucleotide of the domain can comprise a 2′ modification, e.g., a 2-acetylation or a 2′ methylation.

In an embodiment, a method herein involves a second gRNA which is a modular gRNA, e.g., wherein one or more nucleic acid molecules encode a modular gRNA. In other embodiments, the method involves a second gRNA which is a chimeric gRNA. In other embodiments, when the method involves a third or fourth gRNA, the third and fourth gRNA may be a modular gRNA or a chimeric gRNA. When multiple gRNAs are used, any combination of modular or chimeric gRNAs may be used.

Landmarks

Another characteristic of a gRNA molecule is its ability to position a Cas9-mediated cleavage event or break at a desired, e.g., preselected, position on the target nucleic acid. The Cas9-cleavage event can also be characterized as occurring relative to, e.g., within a predefined distance, from a landmark. In an embodiment, one can configure a gRNA such that the gRNA positions a Cas9 molecule so that the Cas9 molecule mediates cleavage, e.g., a double strand or a single strand break, at a preselected position relative to a landmark on a target nucleic acid. In an embodiment, the landmark is the target position, e.g., the nucleotide or one of the nucleotides to be corrected or altered. In an embodiment, the landmark is a position that corresponds to a position in the template nucleic acid, e.g., the 5′ or 3′ end of the replacement sequence, within the replacement sequence, the replacement position where the replacement position is a single nucleotide, the 5′ or 3′ of the template nucleic acid, or the 5′ or 3′ homology arm. In an embodiment, the landmark is an intron/exon boundary, the 5′ or 3′ end or within a coding region, the 5′ or 3′ end or within a transcribed region, or the 5′ or 3′ end or within a repeated element. In an embodiment, the preselected position is at the landmark. In an embodiment, the preselected position is away from the landmark, e.g., within 1, 5, 10, 50, 100, 200, 300, 400, or 500 nucleotides of the landmark, or at least 1, 5, 10, 25, 50 or 100 nucleotides away from the landmark, or 1 to 500, 1 to 400, 1 to 300, 1 to 200, 1 to 100, 10 to 500, 10 to 400, 10 to 300, 10 to 200 or 10 to 100 nucleotides away from the landmark.

II. Methods for Designing gRNAs

Methods for designing gRNAs are described herein, including methods for selecting, designing and validating target domains. Exemplary targeting domains are also provided herein. Targeting Domains discussed herein can be incorporated into the gRNAs described herein.

Methods for selection and validation of target sequences as well as off-target analyses are described, e.g., in Mali et al., 2013 SCIENCE 339(6121): 823-826; Hsu et al. NAT BIOTECHNOL, 31(9): 827-32; Fu et al. (2014) NAT. BIOTECHNOL 32(3): 279-84; Heigwer et al., 2014 NAT METHODS 11(2): 122-3; Bae et al. (2014) BIOINFORMATICS 30(10): 1473-5; Xiao et al. (2014) BIOINFORMATICS 30 (8): 1180-1182. Additional considerations for designing gRNAs are discussed in the section entitled “gRNA Design” in PCT Application WO 2015/048577, the entire contents of which are expressly incorporated herein by reference.

For example, a software tool can be used to optimize the choice of gRNA within a user's target sequence, e.g., to minimize total off-target activity across the genome. Off target activity may be other than cleavage. For each possible gRNA choice using S. pyogenes Cas9, the tool can identify all off-target sequences (preceding either NAG or NGG PAMs) across the genome that contain up to certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base-pairs. The cleavage efficiency at each off-target sequence can be predicted, e.g., using an experimentally-derived weighting scheme. Each possible gRNA is then ranked according to its total predicted off-target cleavage; the top-ranked gRNAs represent those that are likely to have the greatest on-target and the least off-target cleavage. Other functions, e.g., automated reagent design for CRISPR construction, primer design for the on-target Surveyor assay, and primer design for high-throughput detection and quantification of off-target cleavage via next-gen sequencing, can also be included in the tool. Candidate gRNA molecules can be evaluated by art-known methods or as described in Section IV herein.

The targeting domains discussed herein can be incorporated into the gRNAs described herein.

Guide RNAs (gRNAs) for use with S. pyogenes, S. aureus and N. meningitidis Cas9 molecules are identified using a DNA sequence searching algorithm. Guide RNA design is carried out using a custom guide RNA design software based on the public tool cas-offinder (Bae et al. (2014) BIOINFORMATICS 30(10): 1473-5). Said custom guide RNA design software scores guides after calculating their genome-wide off-target propensity. Typically matches ranging from perfect matches to 7 mismatches are considered for guides ranging in length from 17 to 24. Once the off-target sites are computationally determined, an aggregate score is calculated for each guide and summarized in a tabular output using a web-interface. In addition to identifying potential gRNA sites adjacent to PAM sequences, the software also identifies all PAM adjacent sequences that differ by 1, 2, 3 or more nucleotides from the selected gRNA sites. Genomic DNA sequence for each gene was obtained from the UCSC Genome browser and sequences were screened for repeat elements using the publically available RepeatMasker program. RepeatMasker searches input DNA sequences for repeated elements and regions of low complexity. The output is a detailed annotation of the repeats present in a given query sequence.

Following identification, gRNAs are ranked into tiers based on their distance to the target site, their orthogonality and presence of a 5′ G (based on identification of close matches in the human genome containing a relevant PAM (e.g., in the case of S. pyogenes, a NGG PAM, in the case of S. aureus, a NNGRRT or NNGRRV PAM, and in the case of N. meningitidis, a NNNNGATT or NNNNGCTT PAM). Orthogonality refers to the number of sequences in the human genome that contain a minimum number of mismatches to the target sequence. A “high level of orthogonality” or “good orthogonality” may, for example, refer to 20-mer gRNAs that have no identical sequences in the human genome besides the intended target, nor any sequences that contain one or two mismatches in the target sequence. Targeting domains with good orthogonality are selected to minimize off-target DNA cleavage.

gRNAs are identified for both single-gRNA nuclease cleavage and for a dual-gRNA paired “nickase” strategy. Criteria for selecting gRNAs and the determination for which gRNAs can be used for the dual-gRNA paired “nickase” strategy is based on two considerations: gRNA pairs should be oriented on the DNA such that PAMs are facing out and cutting with the D10A Cas9 nickase will result in 5′ overhangs.

An assumption that cleaving with dual nickase pairs will result in deletion of the entire intervening sequence at a reasonable frequency. However, cleaving with dual nickase pairs can also result in indel mutations at the site of only one of the gRNAs. Candidate pair members can be tested for how efficiently they remove the entire sequence versus causing indel mutations at the site of one gRNA.

The targeting domains discussed herein can be incorporated into the gRNAs described herein.

In an embodiment, two or more (e.g., three or four) gRNA molecules are used with one Cas9 molecule. In another embodiment, when two or more (e.g., three or four) gRNAs are used with two or more Cas9 molecules, at least one Cas9 molecule is from a different species than the other Cas9 molecule(s). For example, when two gRNA molecules are used with two Cas9 molecules, one Cas9 molecule can be from one species and the other Cas9 molecule can be from a different species. Both Cas9 species are used to generate a single or double-strand break, as desired.

In some embodiments, the targeting domains described herein are used with a Cas9 nickase molecule to generate a single strand break.

In some embodiments, the targeting domains described herein are used with a Cas9 nuclease molecule to generate a double strand break.

When two gRNAs designed for use to target two Cas9 molecules, one Cas9 can be one species, the second Cas9 can be from a different species. Both Cas9 species are used to generate a single or double-strand break, as desired.

It is contemplated herein that any upstream gRNA described herein may be paired with any downstream gRNA described herein. When an upstream gRNA designed for use with one species of Cas9 is paired with a downstream gRNA designed for use from a different species of Cas9, both Cas9 species are used to generate a single or double-strand break, as desired.

In an embodiment, the targeting domain of a gRNA molecule is configured to avoid unwanted target chromosome elements, such as repeat elements, e.g., Alu elements, in the target domain. The gRNA molecule may be a first, second, third and/or fourth gRNA molecule.

Strategies to Identify HDR-Enhancing gRNAs to Alter a Gene

In some embodiments, the methods described herein comprise altering (e.g., modifying, e.g., by activating or repressing) the expression of a gene (e.g., a gene encoding a protein involved in one or more DNA repair pathways). In some embodiments, the expression of the gene is altered using a HDR-enhancing gRNA. In some embodiments, the methods described herein provide an alteration of (e.g., by repressing) the expression of a gene that does not comprise nucleotide insertion or deletion of the gene. In some embodiments, this type of alteration is also referred to as “knocking down” the expression of the gene.

In other embodiments, the altered expression of a gene, e.g., is mediated by a CRISPR/Cas system comprising a Cas9 molecule (e.g., an eaCas9 molecule or an eiCas9 molecule) and an HDR-enhancing gRNA in order to alter transcription (e.g., to block, reduce, increase transcription, or decrease transcription) of the gene. In some embodiments, where an eiCas9 molecule is used, transcription of the gene is altered temporarily or transiently. In one embodiment, the HDR-enhancing gRNA targets 53BP1, Rif1, PTIP, KU 70, KU 80, XRCC4, XLF, Artemis, BRCA2, BRCA1, CtIP, EXol, DNA2, MRN complex, MRE11, Rad50, NbsI, Rad51, XRCC1, Ligase I, Ligase III, Pol Theta, Fbh1, RTEL, PARI, Rap80, Rad52, ERCC1, XPF, XRCC1, Msh2, Msh3, Msh6, Mlh1, Pms2, or KDM4A/JMJD2A. In another embodiment, the gene may be selected from the group consisting of TP53BP1, RIF1, PAXIP1, XRCC6, XRCC5, PRKDC, LIG4, XRCC4, NHEJ1, DCLRElC, BRCA2, RAD51, XRCC1, LIG1, LIG3, POLQ, FBXO18, RTEL1, PARPBP, UIMC1, RAD52, ERCC1, ERCC4, PARP1, BRCA1, RBBP8, EX01, DNA2, MRE11A, RAD50, NBN, MSH2, MSH3, MSH6, MlH1, PMS2, EZH2, KDM4A/JMJD2A, and CKD1.

In another embodiment, the altered expression of a gene is mediated by a CRISPR/Cas system comprising a Cas9-fusion molecule (e.g., an eiCas9 fusion molecule, e.g., an eiCas9 molecule fused to a transcription repressor domain, a transcription activator domain, or a chromatin modifying domain) and an HDR-enhancing gRNA to alter transcription (e.g., to block, reduce, increase transcription, or decrease transcription) of the gene. In some embodiments, where an eiCas9 molecule is used, transcription of the gene is altered temporarily or transiently. In one embodiment, the HDR-enhancing gRNA targets 53BP1, Rif1, PTIP, KU 70, KU 80, XRCC4, XLF, Artemis, BRCA2, BRCA1, CtIP, EXol, DNA2, MRN complex, MRE11, Rad50, NbsI, Rad51, XRCC1, Ligase I, Ligase III, Pol Theta, Fbh1, RTEL, PARI, Rap80, Rad52, ERCC1, XPF, XRCC1, Msh2, Msh3, Msh6, Mlh1, Pms2, or KDM4A/JMJD2A. In one embodiment, the target gene may be selected from the group consisting of TP53BP1, RIF1, PAXIP1, XRCC6, XRCC5, PRKDC, LIG4, XRCC4, NHEJ1, DCLRElC, BRCA2, RAD51, XRCC1, LIG1, LIG3, POLQ, FBXO18, RTEL1, PARPBP, UIMC1, RAD52, ERCC1, ERCC4, PARP1, BRCA1, RBBP8, EX01, DNA2, MRE11A, RAD50, NBN, MSH2, MSH3, MSH6, MlH1, PMS2, EZH2, KDM4A/JMJD2A, and CKD1.

A transcriptional activator or a transcriptional repressor can be linked, or fused, to any of the Cas9 molecules described herein either covalently or non-covalently. The transcriptional activator or a transcriptional repressor can be linked, covalently or non-covalently, to the N terminus or the C terminus of the Cas9 molecule. The transcriptional activator or a transcriptional repressor can be linked to a residue other than the N or C terminal residue of the Cas9 molecule, e.g., to an internal residue of the Cas9 molecule. In an embodiment the linkage is other than a peptide linkage between amino acid residues of the Cas9/transcriptional activator or a transcriptional repressor, e.g., the linkage is a covalent linkage through a side chain of an amino acid of the Cas 9 molecule and/or the transcriptional activator or a transcriptional repressor. By way of example, the linkage can be a linkage to the terminal N of the side chain of a lysine, e.g., an internal lysine residue, e.g., an inernal lysine residue from any of the Cas 9 domains described herein. In an embodiment the transcriptional activator or a transcriptional repressor is linked, postranslationally, to a Cas 9 molecule. The transcriptional activator or a transcriptional repressor is linked to the Cas9 molecule such that proper folding and function of the Cas9 molecule and the transcriptional activator or a transcriptional repressor is maintained. In an embodiment the linkage is a peptide linkage, e.g., as in a fusion protein.

In an embodiment, a linker, e.g., a linker described herein, is disposed between the Cas9 molecule and the transcriptional activator or a transcriptional repressor. The linker can be disposed at the N terminus of the transcriptional activator or a transcriptional repressor. The linker can be disposed at the C terminus of the transcriptional activator or a transcriptional repressor. In an embodiment, a linker is disposed at the N terminus and the C terminus of the transcriptional activator or a transcriptional repressor. In an embodiment, a linker is disposed between an amino acid residue of the Cas 9 molecule and the transcriptional activator or a transcriptional repressor.

The linker may be a short peptide sequence. Exemplary linkers suitable for use to link a transcriptional activator or a transcriptional repressor to a Cas9 molecule are disclosed herein. In an embodiment, a linker is not used and the Cas9 molecule and the transcriptional activator or a transcriptional repressor are directly linked to each other by a covalent bond, e.g., a peptide bond. In alternative embodiments, the Cas9 molecule and the transcriptional activator or a transcriptional repressor are linked by a covalent bond that is not a peptide bond, e.g., by chemical conjugation.

In an embodiment, the Cas9/transcriptional activator or a transcriptional repressor is a fusion protein, where transcriptional activator or a transcriptional repressor is covalently linked to the Cas9 molecule by a peptide bond. The N terminus or C terminus of the transcriptional activator or a transcriptional repressor can be linked to the N terminus, e.g., the N-terminal residue, or the C terminus, e.g., the C-terminal residue of the Cas9 molecule. In another embodiment, the transcriptional activator or a transcriptional repressor is linked to a residue that is not the N terminal residue or the C terminal residue of the Cas9 molecule, e.g., the transcriptional activator or a transcriptional repressor is linked to an internal residue of the Cas9 molecule. In an embodiment, the transcriptional activator or a transcriptional repressor is inserted to the sequence of a Cas 9 molecule. In an embodiment, the N-terminal residue of the transcriptional activator or a transcriptional repressor is linked to an internal residue of the Cas9 molecule and the C-terminal residue of the transcriptional activator or a transcriptional repressor is linked to an internal residue of the Cas9 molecule.

When the transcriptional activator or a transcriptional repressor is linked to an internal residue of the Cas9 molecule as a fusion protein, the transcriptional activator or a transcriptional repressor is disposed between sequences of the Cas9 molecule, such that the primary structure of the Cas9 fusion protein is organized as follows: Cas9N-L1-transcriptional activator or transcriptional repressor-L2-Cas9C, wherein Cas9N represents an N terminal portion of the sequence of the Cas9 molecule, transcriptional activator or transcriptional repressor represents the transcriptional activator or transcriptional repressor, Cas9C represents a C terminal portion of the Cas9 molecule, L1 is an optional linker, and L2 is an optional linker. A Cas9 fusion protein can comprise L1, L2, or both L1 and L2. L1 and L2 can be the same, or different, e.g., they can differ in length, or in amino acid composition or sequence. In an embodiment the transcriptional activator or transcriptional repressor (with or without L1 and/or L2) can be disposed between two amino acid residues that are adjacent one another in the Cas 9 molecule. In an embodiment the transcriptional activator or transcriptional repressor (with or without L1 and/or L2) can be substituted for one or more amino acid residues of the Cas 9 molecule, e.g., a region of Cas 9 molecule sequence can be deleted and replaced with the transcriptional activator or transcriptional repressor (with or without L1 and/or L2). In an embodiment, a Cas9 fusion protein, comprises a plurality of, e.g., 2, or 3, transcriptional activators or transcriptional repressors (with or without L1 and/or L2).

In an embodiment, a first linker is disposed between Cas9N and the N-terminus of the transcriptional activator or transcriptional repressor and a second linker is disposed between the C-terminus of the transcriptional activator or transcriptional repressor and Cas9C. The linkers disposed between an transcriptional activator or transcriptional repressor and a Cas9, or a portion of a Cas9 molecule, may be selected for specific length and/or flexibility to allow the primary structure of the transcriptional activator or transcriptional repressor and the Cas9 molecule to properly fold such that the transcriptional activator or transcriptional repressor and the Cas9 molecule exhibit functional activity.

In an embodiment, the transcriptional activator or transcriptional repressor is disposed in a region of the Cas9 molecule that is not highly conserved and/or is dispensable for Cas9 activity. For example, the transcriptional activator or transcriptional repressor may be disposed in a REC domain, or in place of all or part of a REC domain. For example, the transcriptional activator or transcriptional repressor (with or without L1 and/or L2) disposed in a REC deletion, e.g., the REC₂ deletion, the REC1_(CT) deletion, or the REC1_(SUB) deletion, as these regions are known for being dispensable for Cas9 activity, and are spatially distant from the regions that mediate Cas9 activity. In this embodiment, when the Cas9/transcriptional activator or transcriptional repressor fusion protein is folded, the regions of the Cas9 molecule, including the regions physically separated by the transcriptional activator or transcriptional repressor sequence in the primary structure, are able to fold such that the Cas9 molecule comprises functional Cas9 activity. In addition, the transcriptional activator or transcriptional repressor is linked to the Cas9 molecule sequences such that the transcriptional activator or transcriptional repressor can also fold such that the transcriptional activator or transcriptional repressor comprises functional transcriptional activator or transcriptional repressor activity.

A fusion protein comprising a Cas9 molecule and a transcriptional activator or transcriptional repressor is generated using standard recombinant DNA techniques known in the art, such as by constructing a recombinant nucleic acid molecule that comprises a nucleic acid sequence encoding the Cas9 molecule and a nucleic acid sequence encoding the transcriptional activator or transcriptional repressor in a configuration such that expression of the recombinant nucleic acid results in production of the Cas9/transcriptional activator or transcriptional repressor fusion protein, e.g., the nucleic acid sequence(s) encoding the Cas9 molecule is in frame with the nucleic acid sequence encoding the transcriptional activator or transcriptional repressor.

In some embodiments, the knockdown of a gene is mediated by a CRISPR/Cas system comprising a Cas9-fusion molecule (e.g., an eiCas9 molecule fused to a transcription repressor domain or a chromatin modifying domain) and an HDR-enhancing gRNA to decrease transcription (e.g., to block, or reduce transcription) of the gene. In some embodiments, the knockdown of a gene is mediated by a CRISPR/Cas system comprising an eiCas9 molecule fused to a transcription repressor domain and an HDR-enhancing gRNA to decrease transcription (e.g., to block, or reduce transcription) of the gene. In some embodiments, where an eiCas9 molecule is used, transcription of the gene is altered temporarily or transiently. In some embodiments, this approach results in a reduction, decrease, repression, or elimination of the expression of the gene (e.g., by inhibiting transcription) of the gene. In some embodiments, the transcription of the target gene is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or greater than 90%, as compared to the level of transcription of the target gene in the absence of the HDR-enhancing gRNA that targets the gene.

In one embodiment, the transcription repressor is KRAB, Mad mSIN3 interaction domain, the ERF repressor domain, a histone lysine methyltransferase (KMT), a histone lysine demthylase (KDM), a histone lysine deacetylase, a DNA methylase, a boundary element, or a periphery recruitment element. In one embodiment, the KMT is SUV39H1, SUV39H2, G9A, Pr-SET7/8, SUV4-10H1, PR-set7, Suv4-20, Set9, EZH2, RIZ1, LSD1/BHC110, SpLsd1/Swm1/Safl10, Su(var)3-3, JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1, JMJD2D, Rph1, JARID1A/RBP2, JARID1B/PLU-1, JARID1C/SMCX, JARID1D/SMCY, Lid, Jhn2, or Jmj2. In one embodiment, the histone lysine deacetylase is HDAC1, HDAC2, HDAC3, HDAC8, Rpd3, Hos1, Cir6, HDAC4, HDAC5, HDAC7, HDAC9, Hdal, Cir3, SIRT1, SIRT2, Sir2, Hst1, Hst2, Hst3, HSt4, or HDAC11. In one embodiment, the DNA methylase is Dam, Dcm, M. SssI, DNMT1, DNMT3a/DNMT3b, METI, DRM3, ZMET2, CMT1, or CMT2. In one embodiment, the boundary element is CTCF. In one embodiment, the periphery recruitment element is Lamin A or Lamin B.

In some embodiments, the altered expression of a gene is mediated by a CRISPR/Cas system comprising a Cas9-fusion molecule (e.g., an eiCas9 molecule fused to a transcription activator domain or a chromatin modifying domain) and an HDR-enhancing gRNA to increase transcription of the gene. In some embodiments, increased expression of a gene is mediated by a CRISPR/Cas system comprising a an eiCas9 molecule fused to a transcription activator domain and an HDR-enhancing gRNA to increase transcription of the gene. In some embodiments, where an eiCas9 molecule is used, transcription of the gene is altered temporarily or transiently. In some embodiments, this approach results in increased expression of the gene (e.g., by increasing transcription) of the gene. In some embodiments, the transcription of the target gene is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 1.1 fold, at least about 1.2 fold, at least about 1.3 fold, at least about 1.4 fold, at least about 1.5 fold, at least about 1.6 fold, at least about 1.7 fold, at least about 1.8 fold, at least about 1.9 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 4.5 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 12 fold, at least about 15 fold, at least about 18 fold, or at least about 20-fold, as compared to the level of transcription of the target gene in the absence of the HDR-enhancing gRNA that targets the gene.

In one embodiment, the transcription activator is GAL4, VP16, VP64, a p65 subdomain (NFkB), a histone lysine methyltransferase (KMT), a histone lysine demethylate (KDM), a histone lysine acetyltransferase (KAT), a DNA demethylase, or a protein docking element. In one embodiment, the KMT is hSET1A, hSET1B, MLL1, MLL2, MLL3, MLL4, MLL5, ASH1, Trx, Trr, Ash1, SYMD2, NSD1, or DOT1. In one embodiment, the KDM is LSD1/BHC110, JHDM2a/b, UTX, or JMJD3. In one embodiment, the KAT is hGCN4, PCAF, dGCN5/PCAF, Gcn5, CBP, p300, dCBP/NEJ, TAF1, TIP60/PLIP, MOZ/MYST3, MORF/MYST4, Mst2, Sas3, CG1894, HBO1/MYST2, CHM, Mst2, HMOF/MYST1, dMOF, Sas2, Mst2, SRC1, ACTR, P160, or CLOCK. In one embodiment, the DNA demethylase is AID, TET1, DME, DML1, DML2, ROS1. In one embodiment, the protein docking element is FKBP/FRB (S. pombe) or Pil1/Abyl (E. coli).

In some embodiments, a non-coding region (e.g., an enhancer region, a promoter region a 5′ UTR, 3′ UTR, and a polyadenylation signal) of a gene is targeted to alter the expression of the gene. In some embodiments, a transcriptional regulatory region, e.g., a promoter region (e.g., a promoter region that controls the transcription of the gene) is targeted to alter (e.g., by knocking-down) the expression of the gene. In certain embodiments, one or more gRNA molecules comprise a targeting domain configured to target an eiCas9 molecule or an eiCas9 fusion protein sufficiently close to the transcriptional regulatory region, e.g., a promoter region (e.g., a promoter region that controls the transcription of the gene) to reduce, decrease or repress expression of the gene.

In some embodiments, the methods described herein provide an alteration of the expression of a gene that does not comprise nucleotide insertion or deletion of the gene. In some embodiments, this approach results in an increase in the expression of the gene. In some embodiments, the increase in expression of a gene is mediated by a CRISPR/Cas system comprising a Cas9 molecule (e.g., an eiCas9 molecule) or a Cas9-fusion molecule (e.g., an eiCas9 fusion molecule (e.g., an eiCas9 molecule fused to a transcription activator domain or a chromatin modifying domain) to alter transcription (e.g., to increase transcription) of the gene. In some embodiments, a non-coding region (e.g., an enhancer region, a promoter region a 5′ UTR, 3′ UTR, and a polyadenylation signal) of a gene is targeted to alter the expression (e.g., increase expression) of the gene. In some embodiments, a transcriptional regulatory region, e.g., a promoter region (e.g., a promoter region that controls the transcription of the gene) is targeted to alter (e.g., by increasing) the expression of the gene. In certain embodiments, one or more gRNA molecules comprise a targeting domain configured to target an eiCas9 molecule or an eiCas9 fusion protein sufficiently close to the transcriptional regulatory region, e.g., a promoter region (e.g., a promoter region that controls the transcription of the gene) to increase expression of the gene.

As an example, three strategies were utilized to identify gRNAs for use with S. pyogenes, S. aureus and N. meningitidis Cas9 molecules. In some embodiments, the identified gRNA may be used to alter (e.g., activate or repress) a gene listed in Table I.

As an example, three strategies were utilized to identify gRNAs for use with S. pyogenes, S. aureus and N. meningitidis Cas9 molecules.

TABLE I Exemplary Genes Targets For Altered Gene Expression Using an HDR-Enhancing gRNA RefSeq Target Name Gene ID TP53BP1 7158 RIF1 55183 PAXIP1 22976 XRCC6 2547 XRCC5 7520 PRKDC 5591 LIG4 3981 XRCC4 7518 NHEJ1 79840 DCLRE1C 64421 BRCA2 675 RAD51 5888 XRCC1 7515 LIG1 3978 LIG3 3980 POLQ 10721 FBXO18 84893 RTEL1 51750 PARPBP 55010 UIMC1 51720 RAD52 5893 ERCC1 2067 ERCC4 2072 PARP1 142 BRCA1 672 RBBP8 5932 EXO1 9156 DNA2 1763 MRE11A 4361 RAD50 10111 NBN 4683 MSH2 4436 MSH3 4437 MSH6 2956 M1H1 4292 PMS2 5395 EZH2 2146 KDM4A/JMJD2A 9682 CDK1 983

As an example, HDR-enhancing gRNAs for use with S. pyogenes, and S. aureus Cas9 molecules were identified using a DNA sequence searching algorithm. HDR-enhancing guide RNA design was carried out using a custom guide RNA design software based on the public tool cas-offinder (Bae et al. (2014)). Said custom guide RNA design software scores guides after calculating their genomewide off-target propensity. Typically matches ranging from perfect matches to 7 mismatches were considered for guides ranging in length from 17 to 24. Once the off-target sites were computationally determined, an aggregate score was calculated for each guide and summarized in a tabular output using a web-interface. In addition to identifying potential gRNA sites adjacent to PAM sequences, the software also identifies all PAM adjacent sequences that differ by 1, 2, 3 or more nucleotides from the selected gRNA sites. Genomic DNA sequence for each gene was obtained from the UCSC Genome browser and sequences were screened for repeat elements using the publically available RepeatMasker program. RepeatMasker searches input DNA sequences for repeated elements and regions of low complexity. The output is a detailed annotation of the repeats present in a given query sequence.

Following identification, HDR-enhancing gRNAs were ranked into tiers based on their distance to the target site, their orthogonality and presence of a 5′ G (based on identification of close matches in the human genome containing a relavant PAM (e.g., in the case of S. pyogenes, a NGG PAM, in the case of S. aureus, a NNGRRT or NNGRRV PAM, and in the case of N. meningitidis, a NNNNGATT or NNNNGCTT PAM). Orthogonality refers to the number of sequences in the human genome that contain a minimum number of mismatches to the target sequence. A “high level of orthogonality” or “good orthogonality” may, for example, refer to 20-mer HDR-enhancing gRNAs that have no identical sequences in the human genome besides the intended target, nor any sequences that contain one or two mismatches in the target sequence. Targeting domains with good orthogonality are selected to minimize off-target DNA cleavage.

For example, for S. pyogenes and N. meningitidis targets, 17-mer, or 20-mer HDR-enhancing gRNAs were designed. As another example, for S. aureus targets, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer and 24-mer HDR-enhancing gRNAs were designed. In some embodiments, the targeting domains, disclosed herein, may comprise the 17-mer described in Tables II.1A, II.1B, II.2A, II.2B, II.3A, II.3B, II.4A, II.4B, II.5A, II.5B, II.6A, II.6B, II.7A, II.7B, II.8A, II.8B, II.9A, II.9B, II.10A, II.10B, II.11A, II.11B, II.12A, II.12B, II.13A, II.13B, II.14A, II.14B, II.15A, II.15B, II.16A, II.16B, II.17A, II.17B, II.18A, II.18B, II.19A, II.19B, II.20A, II.20B, II.21A, II.21B, II.22A, II.22B, II.23A, II.23B, II.24A, II.24B, II.25A, II.25B, II.26A, II.26B, II.27A, II.27B, II.28A, II.28B, II.29A, II.29B, II.30A, II.30B, II.31A, II.31B, II.32A, II.32B, II.33A, II.33B, II.34A, II.34B, II.35A, II.35B, II.36A, II.36B, II.37A, II.37B, II.38A, II.38B, II.39A, II.39B, II.40A, II.40B, II.41A, II.41B, II.42A, II.42B, II.43A, II.43B, II.44A, II.44B, II.45A, II.45B, II.46A, II.46B, II.47A, II.47B, II.48A, II.48B, II.49A, II.49B, II.50A, II.50B, II.51A, II.51B, II.52A, II.52B, II.53A, II.53B, II.54A, II.54B, II.55A, II.55B, II.56A, II.56B, II.57A, II.57B, II.58A, II.58B, II.59A, II.59B, II.60A, II.60B, II.61A, II.61B, II.62A, II.62B, II.63A, II.63B, II.64A, II.64B, II.65A, II.65B, II.66, II.67A, II.67B, II.68A, II.68B, II.69A, II.69B, II.70A, II.70B, II.71A, II.71B, II.72A, II.72B, II.73A, II.73B, II.74A, II.74B, II.75A, II.75B, II.76A, II.76B, II.77A, II.77B, II.78A, or II.78B, e.g., the targeting domains of 18 or more nucleotides may comprise the 17-mer HDR-enhancing gRNAs described in Tables II.1A, II.1B, II.2A, II.2B, II.3A, II.3B, II.4A, II.4B, II.5A, II.5B, II.6A, II.6B, II.7A, II.7B, II.8A, II.8B, II.9A, II.9B, II.10A, II.10B, II.11A, II.11B, II.12A, II.12B, II.13A, II.13B, II.14A, II.14B, II.15A, II.15B, II.16A, II.16B, II.17A, II.17B, II.18A, II.18B, II.19A, II.19B, II.20A, II.20B, II.21A, II.21B, II.22A, II.22B, II.23A, II.23B, II.24A, II.24B, II.25A, II.25B, II.26A, II.26B, II.27A, II.27B, II.28A, II.28B, II.29A, II.29B, II.30A, II.30B, II.31A, II.31B, II.32A, II.32B, II.33A, II.33B, II.34A, II.34B, II.35A, II.35B, II.36A, II.36B, II.37A, II.37B, II.38A, II.38B, II.39A, II.39B, II.40A, II.40B, II.41A, II.41B, II.42A, II.42B, II.43A, II.43B, II.44A, II.44B, II.45A, II.45B, II.46A, II.46B, II.47A, II.47B, II.48A, II.48B, II.49A, II.49B, II.50A, II.50B, II.51A, II.51B, II.52A, II.52B, II.53A, II.53B, II.54A, II.54B, II.55A, II.55B, II.56A, II.56B, II.57A, II.57B, II.58A, II.58B, II.59A, II.59B, II.60A, II.60B, II.61A, II.61B, II.62A, II.62B, II.63A, II.63B, II.64A, II.64B, II.65A, II.65B, II.66, II.67A, II.67B, II.68A, II.68B, II.69A, II.69B, II.70A, II.70B, II.71A, II.71B, II.72A, II.72B, II.73A, II.73B, II.74A, II.74B, II.75A, II.75B, II.76A, II.76B, II.77A, II.77B, II.78A, or II.78B. In some embodiments, the targeting domains, disclosed herein, may comprises the 18-mer described in Tables II.1A, II.1B, II.2A, II.2B, II.3A, II.3B, II.4A, II.4B, II.5A, II.5B, II.6A, II.6B, II.7A, II.7B, II.8A, II.8B, II.9A, II.9B, II.10A, II.10B, II.11A, II.11B, II.12A, II.12B, II.13A, II.13B, II.14A, II.14B, II.15A, II.15B, II.16A, II.16B, II.17A, II.17B, II.18A, II.18B, II.19A, II.19B, II.20A, II.20B, II.21A, II.21B, II.22A, II.22B, II.23A, II.23B, II.24A, II.24B, II.25A, II.25B, II.26A, II.26B, II.27A, II.27B, II.28A, II.28B, II.29A, II.29B, II.30A, II.30B, II.31A, II.31B, II.32A, II.32B, II.33A, II.33B, II.34A, II.34B, II.35A, II.35B, II.36A, II.36B, II.37A, II.37B, II.38A, II.38B, II.39A, II.39B, II.40A, II.40B, II.41A, II.41B, II.42A, II.42B, II.43A, II.43B, II.44A, II.44B, II.45A, II.45B, II.46A, II.46B, II.47A, II.47B, II.48A, II.48B, II.49A, II.49B, II.50A, II.50B, II.51A, II.51B, II.52A, II.52B, II.53A, II.53B, II.54A, II.54B, II.55A, II.55B, II.56A, II.56B, II.57A, II.57B, II.58A, II.58B, II.59A, II.59B, II.60A, II.60B, II.61A, II.61B, II.62A, II.62B, II.63A, II.63B, II.64A, II.64B, II.65A, II.65B, II.66, II.67A, II.67B, II.68A, II.68B, II.69A, II.69B, II.70A, II.70B, II.71A, II.71B, II.72A, II.72B, II.73A, II.73B, II.74A, II.74B, II.75A, II.75B, II.76A, II.76B, II.77A, II.77B, II.78A, or II.78B, e.g., the targeting domains of 19 or more nucleotides may comprise the 18-mer HDR-enhancing gRNAs described in Tables II.1A, II.1B, II.2A, II.2B, II.3A, II.3B, II.4A, II.4B, II.5A, II.5B, II.6A, II.6B, II.7A, II.7B, II.8A, II.8B, II.9A, II.9B, II.10A, II.10B, II.11A, II.11B, II.12A, II.12B, II.13A, II.13B, II.14A, II.14B, II.15A, II.15B, II.16A, II.16B, II.17A, II.17B, II.18A, II.18B, II.19A, II.19B, II.20A, II.20B, II.21A, II.21B, II.22A, II.22B, II.23A, II.23B, II.24A, II.24B, II.25A, II.25B, II.26A, II.26B, II.27A, II.27B, II.28A, II.28B, II.29A, II.29B, II.30A, II.30B, II.31A, II.31B, II.32A, II.32B, II.33A, II.33B, II.34A, II.34B, II.35A, II.35B, II.36A, II.36B, II.37A, II.37B, II.38A, II.38B, II.39A, II.39B, II.40A, II.40B, II.41A, II.41B, II.42A, II.42B, II.43A, II.43B, II.44A, II.44B, II.45A, II.45B, II.46A, II.46B, II.47A, II.47B, II.48A, II.48B, II.49A, II.49B, II.50A, II.50B, II.51A, II.51B, II.52A, II.52B, II.53A, II.53B, II.54A, II.54B, II.55A, II.55B, II.56A, II.56B, II.57A, II.57B, II.58A, II.58B, II.59A, II.59B, II.60A, II.60B, II.61A, II.61B, II.62A, II.62B, II.63A, II.63B, II.64A, II.64B, II.65A, II.65B, II.66, II.67A, II.67B, II.68A, II.68B, II.69A, II.69B, II.70A, II.70B, II.71A, II.71B, II.72A, II.72B, II.73A, II.73B, II.74A, II.74B, II.75A, II.75B, II.76A, II.76B, II.77A, II.77B, II.78A, or II.78B. In some embodiments, the targeting domains, disclosed herein, may comprises the 19-mer described in Tables II.1A, II.1B, II.2A, II.2B, II.3A, II.3B, II.4A, II.4B, II.5A, II.5B, II.6A, II.6B, II.7A, II.7B, II.8A, II.8B, II.9A, II.9B, II.10A, II.10B, II.11A, II.11B, II.12A, II.12B, II.13A, II.13B, II.14A, II.14B, II.15A, II.15B, II.16A, II.16B, II.17A, II.17B, II.18A, II.18B, II.19A, II.19B, II.20A, II.20B, II.21A, II.21B, II.22A, II.22B, II.23A, II.23B, II.24A, II.24B, II.25A, II.25B, II.26A, II.26B, II.27A, II.27B, II.28A, II.28B, II.29A, II.29B, II.30A, II.30B, II.31A, II.31B, II.32A, II.32B, II.33A, II.33B, II.34A, II.34B, II.35A, II.35B, II.36A, II.36B, II.37A, II.37B, II.38A, II.38B, II.39A, II.39B, II.40A, II.40B, II.41A, II.41B, II.42A, II.42B, II.43A, II.43B, II.44A, II.44B, II.45A, II.45B, II.46A, II.46B, II.47A, II.47B, II.48A, II.48B, II.49A, II.49B, II.50A, II.50B, II.51A, II.51B, II.52A, II.52B, II.53A, II.53B, II.54A, II.54B, II.55A, II.55B, II.56A, II.56B, II.57A, II.57B, II.58A, II.58B, II.59A, II.59B, II.60A, II.60B, II.61A, II.61B, II.62A, II.62B, II.63A, II.63B, II.64A, II.64B, II.65A, II.65B, II.66, II.67A, II.67B, II.68A, II.68B, II.69A, II.69B, II.70A, II.70B, II.71A, II.71B, II.72A, II.72B, II.73A, II.73B, II.74A, II.74B, II.75A, II.75B, II.76A, II.76B, II.77A, II.77B, II.78A, or II.78B, e.g., the targeting domains of 20 or more nucleotides may comprise the 19-mer HDR-enhancing gRNAs described in Tables II.1A, II.1B, II.2A, II.2B, II.3A, II.3B, II.4A, II.4B, II.5A, II.5B, II.6A, II.6B, II.7A, II.7B, II.8A, II.8B, II.9A, II.9B, II.10A, II.10B, II.11A, II.11B, II.12A, II.12B, II.13A, II.13B, II.14A, II.14B, II.15A, II.15B, II.16A, II.16B, II.17A, II.17B II.18A, II.18B, II.19A, II.19B, II.20A, II.20B, II.21A, II.21B, II.22A, II.22B, II.23A, II.23B, II.24A, II.24B, II.25A, II.25B, II.26A, II.26B, II.27A, II.27B, II.28A, II.28B, II.29A, II.29B, II.30A, II.30B, II.31A, II.31B, II.32A, II.32B, II.33A, II.33B, II.34A, II.34B, II.35A, II.35B, II.36A, II.36B, II.37A, II.37B, II.38A, II.38B, II.39A, II.39B, II.40A, II.40B, II.41A, II.41B, II.42A, II.42B, II.43A, II.43B, II.44A, II.44B, II.45A, II.45B, II.46A, II.46B, II.47A, II.47B, II.48A, II.48B, II.49A, II.49B, II.50A, II.50B, II.51A, II.51B, II.52A, II.52B, II.53A, II.53B, II.54A, II.54B, II.55A, II.55B, II.56A, II.56B, II.57A, II.57B, II.58A, II.58B, II.59A, II.59B, II.60A, II.60B, II.61A, II.61B, II.62A, II.62B, II.63A, II.63B, II.64A, II.64B, II.65A, II.65B, II.66, II.67A, II.67B, II.68A, II.68B, II.69A, II.69B, II.70A, II.70B, II.71A, II.71B, II.72A, II.72B, II.73A, II.73B, II.74A, II.74B, II.75A, II.75B, II.76A, II.76B, II.77A, II.77B, II.78A, or II.78B. In some embodiments, the targeting domains, disclosed herein, may comprises the 20-mer gRNAs described in Tables II.1A, II.1B, II.2A, II.2B, II.3A, II.3B, II.4A, II.4B, II.5A, II.5B, II.6A, II.6B, II.7A, II.7B, II.8A, II.8B, II.9A, II.9B, II.10A, II.10B, II.11A, II.11B, II.12A, II.12B, II.13A, II.13B, II.14A, II.14B, II.15A, II.15B, II.16A, II.16B, II.17A, II.17B, II.18A, II.18B, II.19A, II.19B, II.20A, II.20B, II.21A, II.21B, II.22A, II.22B, II.23A, II.23B, II.24A, II.24B, II.25A, II.25B, II.26A, II.26B, II.27A, II.27B, II.28A, II.28B, II.29A, II.29B, II.30A, II.30B, II.31A, II.31B, II.32A, II.32B, II.33A, II.33B, II.34A, II.34B, II.35A, II.35B, II.36A, II.36B, II.37A, II.37B, II.38A, II.38B, II.39A, II.39B, II.40A, II.40B, II.41A, II.41B, II.42A, II.42B, II.43A, II.43B, II.44A, II.44B, II.45A, II.45B, II.46A, II.46B, II.47A, II.47B, II.48A, II.48B, II.49A, II.49B, II.50A, II.50B, II.51A, II.51B, II.52A, II.52B, II.53A, II.53B, II.54A, II.54B, II.55A, II.55B, II.56A, II.56B, II.57A, II.57B, II.58A, II.58B, II.59A, II.59B, II.60A, II.60B, II.61A, II.61B, II.62A, II.62B, II.63A, II.63B, II.64A, II.64B, II.65A, II.65B, II.66, II.67A, II.67B, II.68A, II.68B, II.69A, II.69B, II.70A, II.70B, II.71A, II.71B, II.72A, II.72B, II.73A, II.73B, II.74A, II.74B, II.75A, II.75B, II.76A, II.76B, II.77A, II.77B, II.78A, or II.78B, e.g., the targeting domains of 21 or more nucleotides may comprise the 20-mer HDR-enhancing gRNAs described in Tables II.1A, II.1B, II.2A, II.2B, II.3A, II.3B, II.4A, II.4B, II.5A, II.5B, II.6A, II.6B, II.7A, II.7B, II.8A, II.8B, II.9A, II.9B, II.10A, II.10B, II.11A, II.11B, II.12A, II.12B, II.13A, II.13B, II.14A, II.14B, II.15A, II.15B, II.16A, II.16B, II.17A, II.17B, II.18A, II.18B, II.19A, II.19B, II.20A, II.20B, II.21A, II.21B, II.22A, II.22B, II.23A, II.23B, II.24A, II.24B, II.25A, II.25B, II.26A, II.26B, II.27A, II.27B, II.28A, II.28B, II.29A, II.29B, II.30A, II.30B, II.31A, II.31B, II.32A, II.32B, II.33A, II.33B, II.34A, II.34B, II.35A, II.35B, II.36A, II.36B, II.37A, II.37B, II.38A, II.38B, II.39A, II.39B, II.40A, II.40B, II.41A, II.41B, II.42A, II.42B, II.43A, II.43B, II.44A, II.44B, II.45A, II.45B, II.46A, II.46B, II.47A, II.47B, II.48A, II.48B, II.49A, II.49B, II.50A, II.50B, II.51A, II.51B, II.52A, II.52B, II.53A, II.53B, II.54A, II.54B, II.55A, II.55B, II.56A, II.56B, II.57A, II.57B, II.58A, II.58B, II.59A, II.59B, II.60A, II.60B, II.61A, II.61B, II.62A, II.62B, II.63A, II.63B, II.64A, II.64B, II.65A, II.65B, II.66, II.67A, II.67B, II.68A, II.68B, II.69A, II.69B, II.70A, II.70B, II.71A, II.71B, II.72A, II.72B, II.73A, II.73B, II.74A, II.74B, II.75A, II.75B, II.76A, II.76B, II.77A, II.77B, II.78A, or II.78B. In some embodiments, the targeting domains, disclosed herein, may comprises the 21-mer described in Tables II.1A, II.1B, II.2A, II.2B, II.3A, II.3B, II.4A, II.4B, II.5A, II.5B, II.6A, II.6B, II.7A, II.7B, II.8A, II.8B, II.9A, II.9B, II.10A, II.10B, II.11A, II.11B, II.12A, II.12B, II.13A, II.13B, II.14A, II.14B, II.15A, II.15B, II.16A, II.16B, II.17A, II.17B, II.18A, II.18B, II.19A, II.19B, II.20A, II.20B, II.21A, II.21B, II.22A, II.22B, II.23A, II.23B, II.24A, II.24B, II.25A, II.25B, II.26A, II.26B, II.27A, II.27B, II.28A, II.28B, II.29A, II.29B, II.30A, II.30B, II.31A, II.31B, II.32A, II.32B, II.33A, II.33B, II.34A, II.34B, II.35A, II.35B, II.36A, II.36B, II.37A, II.37B, II.38A, II.38B, II.39A, II.39B, II.40A, II.40B, II.41A, II.41B, II.42A, II.42B, II.43A, II.43B, II.44A, II.44B, II.45A, II.45B, II.46A, II.46B, II.47A, II.47B, II.48A, II.48B, II.49A, II.49B, II.50A, II.50B, II.51A, II.51B, II.52A, II.52B, II.53A, II.53B, II.54A, II.54B, II.55A, II.55B, II.56A, II.56B, II.57A, II.57B, II.58A, II.58B, II.59A, II.59B, II.60A, II.60B, II.61A, II.61B, II.62A, II.62B, II.63A, II.63B, II.64A, II.64B, II.65A, II.65B, II.66, II.67A, II.67B, II.68A, II.68B, II.69A, II.69B, II.70A, II.70B, II.71A, II.71B, II.72A, II.72B, II.73A, II.73B, II.74A, II.74B, II.75A, II.75B, II.76A, II.76B, II.77A, II.77B, II.78A, or II.78B, e.g., the targeting domains of 22 or more nucleotides may comprise the 21-mer HDR-enhancing gRNAs described in Tables II.1A, II.1B, II.2A, II.2B, II.3A, II.3B, II.4A, II.4B, II.5A, II.5B, II.6A, II.6B, II.7A, II.7B, II.8A, II.8B, II.9A, II.9B, II.10A, II.10B, II.11A, II.11B, II.12A, II.12B, II.13A, II.13B, II.14A, II.14B, II.15A, II.15B, II.16A, II.16B, II.17A, II.17B, II.18A, II.18B, II.19A, II.19B, II.20A, II.20B, II.21A, II.21B, II.22A, II.22B, II.23A, II.23B, II.24A, II.24B, II.25A, II.25B, II.26A, II.26B, II.27A, II.27B, II.28A, II.28B, II.29A, II.29B, II.30A, II.30B, II.31A, II.31B, II.32A, II.32B, II.33A, II.33B, II.34A, II.34B, II.35A, II.35B, II.36A, II.36B, II.37A, II.37B, II.38A, II.38B, II.39A, II.39B, II.40A, II.40B, II.41A, II.41B, II.42A, II.42B, II.43A, II.43B, II.44A, II.44B, II.45A, II.45B, II.46A, II.46B, II.47A, II.47B, II.48A, II.48B, II.49A, II.49B, II.50A, II.50B, II.51A, II.51B, II.52A, II.52B, II.53A, II.53B, II.54A, II.54B, II.55A, II.55B, II.56A, II.56B, II.57A, II.57B, II.58A, II.58B, II.59A, II.59B, II.60A, II.60B, II.61A, II.61B, II.62A, II.62B, II.63A, II.63B, II.64A, II.64B, II.65A, II.65B, II.66, II.67A, II.67B, II.68A, II.68B, II.69A, II.69B, II.70A, II.70B, II.71A, II.71B, II.72A, II.72B, II.73A, II.73B, II.74A, II.74B, II.75A, II.75B, II.76A, II.76B, II.77A, II.77B, II.78A, or II.78B. In some embodiments, the targeting domains, disclosed herein, may comprises the 22-mer described in Tables II.1A, II.1B, II.2A, II.2B, II.3A, II.3B, II.4A, II.4B, II.5A, II.5B, II.6A, II.6B, II.7A, II.7B, II.8A, II.8B, II.9A, II.9B, II.10A, II.10B, II.11A, II.11B, II.12A, II.12B, II.13A, II.13B, II.14A, II.14B, II.15A, II.15B, II.16A, II.16B, II.17A, II.17B, II.18A, II.18B, II.19A, II.19B, II.20A, II.20B, II.21A, II.21B, II.22A, II.22B, II.23A, II.23B, II.24A, II.24B, II.25A, II.25B, II.26A, II.26B, II.27A, II.27B, II.28A, II.28B, II.29A, II.29B, II.30A, II.30B, II.31A, II.31B, II.32A, II.32B, II.33A, II.33B, II.34A, II.34B, II.35A, II.35B, II.36A, II.36B, II.37A, II.37B, II.38A, II.38B, II.39A, II.39B, II.40A, II.40B, II.41A, II.41B, II.42A, II.42B, II.43A, II.43B, II.44A, II.44B, II.45A, II.45B, II.46A, II.46B, II.47A, II.47B, II.48A, II.48B, II.49A, II.49B, II.50A, II.50B, II.51A, II.51B, II.52A, II.52B, II.53A, II.53B, II.54A, II.54B, II.55A, II.55B, II.56A, II.56B, II.57A, II.57B, II.58A, II.58B, II.59A, II.59B, II.60A, II.60B, II.61A, II.61B, II.62A, II.62B, II.63A, II.63B, II.64A, II.64B, II.65A, II.65B, II.66, II.67A, II.67B, II.68A, II.68B, II.69A, II.69B, II.70A, II.70B, II.71A, II.71B, II.72A, II.72B, II.73A, II.73B, II.74A, II.74B, II.75A, II.75B, II.76A, II.76B, II.77A, II.77B, II.78A, or II.78B, e.g., the targeting domains of 23 or more nucleotides may comprise the 22-mer HDR-enhancing gRNAs described in Tables II.1A, II.1B, II.2A, II.2B, II.3A, II.3B, II.4A, II.4B, II.5A, II.5B, II.6A, II.6B, II.7A, II.7B, II.8A, II.8B, II.9A, II.9B, II.10A, II.10B, II.11A, II.11B, II.12A, II.12B, II.13A, II.13B, II.14A, II.14B, II.15A, II.15B, II.16A, II.16B, II.17A, II.17B, II.18A, II.18B, II.19A, II.19B, II.20A, II.20B, II.21A, II.21B, II.22A, II.22B, II.23A, II.23B, II.24A, II.24B, II.25A, II.25B, II.26A, II.26B, II.27A, II.27B, II.28A, II.28B, II.29A, II.29B, II.30A, II.30B, II.31A, II.31B, II.32A, II.32B, II.33A, II.33B, II.34A, II.34B, II.35A, II.35B, II.36A, II.36B, II.37A, II.37B, II.38A, II.38B, II.39A, II.39B, II.40A, II.40B, II.41A, II.41B, II.42A, II.42B, II.43A, II.43B, II.44A, II.44B, II.45A, II.45B, II.46A, II.46B, II.47A, II.47B, II.48A, II.48B, II.49A, II.49B, II.50A, II.50B, II.51A, II.51B, II.52A, II.52B, II.53A, II.53B, II.54A, II.54B, II.55A, II.55B, II.56A, II.56B, II.57A, II.57B, II.58A, II.58B, II.59A, II.59B, II.60A, II.60B, II.61A, II.61B, II.62A, II.62B, II.63A, II.63B, II.64A, II.64B, II.65A, II.65B, II.66, II.67A, II.67B, II.68A, II.68B, II.69A, II.69B, II.70A, II.70B, II.71A, II.71B, II.72A, II.72B, II.73A, II.73B, II.74A, II.74B, II.75A, II.75B, II.76A, II.76B, II.77A, II.77B, II.78A, or II.78B. In some embodiments, the targeting domains, disclosed herein, may comprises the 23-mer described in Tables II.1A, II.1B, II.2A, II.2B, II.3A, II.3B, II.4A, II.4B, II.5A, II.5B, II.6A, II.6B, II.7A, II.7B, II.8A, II.8B, II.9A, II.9B, II.10A, II.10B, II.11A, II.11B, II.12A, II.12B, II.13A, II.13B, II.14A, II.14B, II.15A, II.15B, II.16A, II.16B, II.17A, II.17B, II.18A, II.18B, II.19A, II.19B, II.20A, II.20B, II.21A, II.21B, II.22A, II.22B, II.23A, II.23B, II.24A, II.24B, II.25A, II.25B, II.26A, II.26B, II.27A, II.27B, II.28A, II.28B, II.29A, II.29B, II.30A, II.30B, II.31A, II.31B, II.32A, II.32B, II.33A, II.33B, II.34A, II.34B, II.35A, II.35B, II.36A, II.36B, II.37A, II.37B, II.38A, II.38B, II.39A, II.39B, II.40A, II.40B, II.41A, II.41B, II.42A, II.42B, II.43A, II.43B, II.44A, II.44B, II.45A, II.45B, II.46A, II.46B, II.47A, II.47B, II.48A, II.48B, II.49A, II.49B, II.50A, II.50B, II.51A, II.51B, II.52A, II.52B, II.53A, II.53B, II.54A, II.54B, II.55A, II.55B, II.56A, II.56B, II.57A, II.57B, II.58A, II.58B, II.59A, II.59B, II.60A, II.60B, II.61A, II.61B, II.62A, II.62B, II.63A, II.63B, II.64A, II.64B, II.65A, II.65B, II.66, II.67A, II.67B, II.68A, II.68B, II.69A, II.69B, II.70A, II.70B, II.71A, II.71B, II.72A, II.72B, II.73A, II.73B, II.74A, II.74B, II.75A, II.75B, II.76A, II.76B, II.77A, II.77B, II.78A, or II.78B, e.g., the targeting domains of 24 or more nucleotides may comprise the 23-mer HDR-enhancing gRNAs described in Tables II.1A, II.1B, II.2A, II.2B, II.3A, II.3B, II.4A, II.4B, II.5A, II.5B, II.6A, II.6B, II.7A, II.7B, II.8A, II.8B, II.9A, II.9B, II.10A, II.10B, II.11A, II.11B, II.12A, II.12B, II.13A, II.13B, II.14A, II.14B, II.15A, II.15B, II.16A, II.16B, II.17A, II.17B, II.18A, II.18B, II.19A, II.19B, II.20A, II.20B, II.21A, II.21B, II.22A, II.22B, II.23A, II.23B, II.24A, II.24B, II.25A, II.25B, II.26A, II.26B, II.27A, II.27B, II.28A, II.28B, II.29A, II.29B, II.30A, II.30B, II.31A, II.31B, II.32A, II.32B, II.33A, II.33B, II.34A, II.34B, II.35A, II.35B, II.36A, II.36B, II.37A, II.37B, II.38A, II.38B, II.39A, II.39B, II.40A, II.40B, II.41A, II.41B, II.42A, II.42B, II.43A, II.43B, II.44A, II.44B, II.45A, II.45B, II.46A, II.46B, II.47A, II.47B, II.48A, II.48B, II.49A, II.49B, II.50A, II.50B, II.51A, II.51B, II.52A, II.52B, II.53A, II.53B, II.54A, II.54B, II.55A, II.55B, II.56A, II.56B, II.57A, II.57B, II.58A, II.58B, II.59A, II.59B, II.60A, II.60B, II.61A, II.61B, II.62A, II.62B, II.63A, II.63B, II.64A, II.64B, II.65A, II.65B, II.66, II.67A, II.67B, II.68A, II.68B, II.69A, II.69B, II.70A, II.70B, II.71A, II.71B, II.72A, II.72B, II.73A, II.73B, II.74A, II.74B, II.75A, II.75B, II.76A, II.76B, II.77A, II.77B, II.78A, or II.78B. In some embodiments, the targeting domains, disclosed herein, may comprises the 24-mer described in Tables II.1A, II.1B, II.2A, II.2B, II.3A, II.3B, II.4A, II.4B, II.5A, II.5B, II.6A, II.6B, II.7A, II.7B, II.8A, II.8B, II.9A, II.9B, II.10A, II.10B, II.11A, II.11B, II.12A, II.12B, II.13A, II.13B, II.14A, II.14B, II.15A, II.15B, II.16A, II.16B, II.17A, II.17B II.18A, II.18B, II.19A, II.19B, II.20A, II.20B, II.21A, II.21B, II.22A, II.22B, II.23A, II.23B, II.24A, II.24B, II.25A, II.25B, II.26A, II.26B, II.27A, II.27B, II.28A, II.28B, II.29A, II.29B, II.30A, II.30B, II.31A, II.31B, II.32A, II.32B, II.33A, II.33B, II.34A, II.34B, II.35A, II.35B, II.36A, II.36B, II.37A, II.37B, II.38A, II.38B, II.39A, II.39B, II.40A, II.40B, II.41A, II.41B, II.42A, II.42B, II.43A, II.43B, II.44A, II.44B, II.45A, II.45B, II.46A, II.46B, II.47A, II.47B, II.48A, II.48B, II.49A, II.49B, II.50A, II.50B, II.51A, II.51B, II.52A, II.52B, II.53A, II.53B, II.54A, II.54B, II.55A, II.55B, II.56A, II.56B, II.57A, II.57B, II.58A, II.58B, II.59A, II.59B, II.60A, II.60B, II.61A, II.61B, II.62A, II.62B, II.63A, II.63B, II.64A, II.64B, II.65A, II.65B, II.66, II.67A, II.67B, II.68A, II.68B, II.69A, II.69B, II.70A, II.70B, II.71A, II.71B, II.72A, II.72B, II.73A, II.73B, II.74A, II.74B, II.75A, II.75B, II.76A, II.76B, II.77A, II.77B, II.78A, or II.78B, e.g., the targeting domains of 25 or more nucleotides may comprise the 24-mer HDR-enhancing gRNAs described in Tables II.1A, II.1B, II.2A, II.2B, II.3A, II.3B, II.4A, II.4B, II.5A, II.5B, II.6A, II.6B, II.7A, II.7B, II.8A, II.8B, II.9A, II.9B, II.10A, II.10B, II.11A, II.11B, II.12A, II.12B, II.13A, II.13B, II.14A, II.14B, II.15A, II.15B, II.16A, II.16B, II.17A, II.17B, II.18A, II.18B, II.19A, II.19B, II.20A, II.20B, II.21A, II.21B, II.22A, II.22B, II.23A, II.23B, II.24A, II.24B, II.25A, II.25B, II.26A, II.26B, II.27A, II.27B, II.28A, II.28B, II.29A, II.29B, II.30A, II.30B, II.31A, II.31B, II.32A, II.32B, II.33A, II.33B, II.34A, II.34B, II.35A, II.35B, II.36A, II.36B, II.37A, II.37B, II.38A, II.38B, II.39A, II.39B, II.40A, II.40B, II.41A, II.41B, II.42A, II.42B, II.43A, II.43B, II.44A, II.44B, II.45A, II.45B, II.46A, II.46B, II.47A, II.47B, II.48A, II.48B, II.49A, II.49B, II.50A, II.50B, II.51A, II.51B, II.52A, II.52B, II.53A, II.53B, II.54A, II.54B, II.55A, II.55B, II.56A, II.56B, II.57A, II.57B, II.58A, II.58B, II.59A, II.59B, II.60A, II.60B, II.61A, II.61B, II.62A, II.62B, II.63A, II.63B, II.64A, II.64B, II.65A, II.65B, II.66, II.67A, II.67B, II.68A, II.68B, II.69A, II.69B, II.70A, II.70B, II.71A, II.71B, II.72A, II.72B, II.73A, II.73B, II.74A, II.74B, II.75A, II.75B, II.76A, II.76B, II.77A, II.77B, II.78A, or II.78B.

The targeting domains discussed herein can be incorporated into any of the HDR-enhancing gRNAs described herein.

HDR-enhancing gRNAs were identified and ranked into 4 tiers for S. pyogenes (Tables II.1A, II.1B, II.3A, II.3B, II.5A, II.5B, II.7A, II.7B, II.9A, II.9B, II.11A, II.11B, II.13A, II.13B, II.15A, II.15B, II.17A, II.17B, II.19A, II.19B, II.21A, II.21B, II.23A, II.23B, II.25A, II.25B, II.27A, II.27B, II.29A, II.29B, II.31A, II.31B, II.33A, II.33B, II.35A, II.35B, II.37A, II.37B, II.39A, II.39B, II.41A, II.41B, II.43A, II.43B, II.45A, II.45B, II.47A, II.47B, II.49A, II.49B, II.51A, II.51B, II.53A, II.53B, II.55A, II.55B, II.57A, II.57B, II.59A, II.59B, II.61A, II.61B, II.63A, II.63B, II.65A, II.65B, II.67A, II.67B, II.69A, II.69B, II.71A, II.71B, II.73A, II.73B, II.75A, II.75B, II.77A, II.77B) and 5 tiers for S. aureus (Tables II.2A, II.2B, II.4A, II.4B, II.6A, II.6B, II.8A, II.8B, II.10A, II.10B, II.12A, II.12B, II.14A, II.14B, II.16A, II.16B, II.18A, II.18B, II.20A, II.20B, II.22A, II.22B, II.24A, II.24B, II.26A, II.26B, II.28A, II.28B, II.30A, II.30B, II.32A, II.32B, II.34A, II.34B, II.36A, II.36B, II.38A, II.38B, II.40A, II.40B, II.42A, II.42B, II.44A, II.44B, II.46A, II.46B, II.48A, II.48B, II.50A, II.50B, II.52A, II.52B, II.54A, II.54B, II.56A, II.56B, II.58A, II.58B, II.60A, II.60B, II.62A, II.62B, II.64A, II.64B, II.66, II.68A, II.68B, II.70A, II.70B, II.72A, II.72B, II.74A, II.74B, II.76A, II.76B, II.78A, or II.78B).

For S. pyogenes, the targeting domains for tier 1 HDR-enhancing gRNA molecules were selected based on (1) distance to a target site (e.g., within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, (2) a high level of orthogonality and (3) the presence of 5′G. The targeting domain for tier 2 HDR-enhancing gRNA molecules were selected based on (1) distance to a target site (e.g., within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and (2) a high level of orthogonality. The targeting domain for tier 3 HDR-enhancing gRNA molecules were selected based on (1) distance to a target site (e.g., within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and (2) the presence of 5′G. The targeting domain for tier 4 HDR-enhancing gRNA molecules were selected based on distance to a target site (e.g., within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS.

For S. aureus, the targeting domain for tier 1 HDR-enhancing gRNA molecules were selected based on (1) distance to a target site (e.g., within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, (2) a high level of orthogonality, (3) the presence of 5′G and (4) PAM is NNGRRT. The targeting domain for tier HDR-enhancing 2 gRNA molecules were selected based on (1) distance to a target site (e.g., within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, (2) a high level of orthogonality, and (3) PAM is NNGRRT. The targeting domain for tier 3 HDR-enhancing gRNA molecules were selected based on (1) distance to a target site (e.g., within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and (2) PAM is NNGRRT. The targeting domain for tier 4 HDR-enhancing gRNA molecules were selected based on (1) distance to a target site (e.g., within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and (2) PAM is NNGRRT. The targeting domain for tier 5 HDR-enhancing gRNA molecules were selected based on (1) distance to a target site (e.g., within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and (2) PAM is NNGRRV.

Note that tiers are non-inclusive (each HDR-enhancing gRNA is listed only once for the strategy). In some instances, no HDR-enhancing gRNA was identified based on the criteria of the particular tier.

Exemplary HDR-enhancing gRNAs targeting the genes listed in Table I are listed in Tables II.2A-II.78B.

Table II.1A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., TP53BP1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the TP53BP1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the TP53BP1 gene.

TABLE II.1A Exemplary HDR-enhancing gRNAs Targeting a TP53B1 Gene A high level of orthogonality, and starts with a G S. pyogenes 1A 1st Tier Target gRNA DNA Site Seq Name Strand Targeting Domain Length ID TP53BP1-1 + GACCUAGGGAUCGAUCUGGA 20 387 TP53BP1-2 + GACCUCUAGCUCGAGCGCGA 20 388 TP53BP1-3 + GACGGGAAAGGGGGAGUUCG 20 389 TP53BP1-4 + GAGCGCGAGGGACCUCCCGC 20 390 TP53BP1-5 + GAGUUCGCGGCCGGUGGCGG 20 391 TP53BP1-6 + GAUCGAUCUGGAGGGACUUG 20 392 TP53BP1-7 − GCUGUCGCCACCGCCGCCAC 20 393 TP53BP1-8 + GGAAAGGGGGAGUUCGCGGC 20 394 TP53BP1-9 + GGACCUCCCGCCGGGAUGCC 20 395 TP53BP1-10 + GGAUCGAUCUGGAGGGACUU 20 396 TP53BP1-11 + GGGAUCGAUCUGGAGGGACU 20 397 TP53BP1-12 + GGGAUUUCUUGAGUGGCGGG 20 398 TP53BP1-13 + GGGGAGUUCGCGGCCGGUGG 20 399 TP53BP1-14 + GGUACUGUUUGGAGAGAAAU 20 400 TP53BP1-15 + GGUGGCGACAGCGGCGACCU 20 401 TP53BP1-16 − GUACCAGGCAUCCCGGCGGG 20 402 TP53BP1-17 − GUCCCUCCAGAUCGAUCCCU 20 403 TP53BP1-18 − GUCCCUCGCGCUCGAGCUAG 20 404 TP53BP1-19 + GUGGCGACAGCGGCGACCUA 20 405 TP53BP1-20 + GUGUGACGUGACGGGAAAGG 20 406

Table II.11 provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., TP53BP1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the TP53BP1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the TP53BP1 gene.

TABLE II.1B Exemplary HDR-enhancing gRNAs Targeting a TP53B1 Gene A high level of orthogonality 1B S. pyogenes Target 2nd Tier DNA Site Seq gRNA Name Strand Targeting Domain Length ID TP53BP1-21 − AACAGUACCAGGCAUCCCGG 20 407 TP53BP1-22 + AAGGGGGAGUUCGCGGCCGG 20 408 TP53BP1-23 − ACAGUACCAGGCAUCCCGGC 20 409 TP53BP1-24 + AGACCUCUAGCUCGAGCGCG 20 410 TP53BP1-25 + AGCGCGAGGGACCUCCCGCC 20 411 TP53BP1-26 + AUUGUGUGACGUGACGGGAA 20 412 TP53BP1-27 + AUUUCUUGAGUGGCGGGCGG 20 413 TP53BP1-28 − CAAGAAAUCCCGUGGAUGAU 20 414 TP53BP1-29 + CAUCCACGGGAUUUCUUGAG 20 415 TP53BP1-30 − CCCGUCACGUCACACAAUAU 20 416 TP53BP1-31 + CCGAUAUUGUGUGACGUGAC 20 417 TP53BP1-32 + CCGCAGCUACCUAUCAUCCA 20 418 TP53BP1-33 − CCGCCACUCAAGAAAUCCCG 20 419 TP53BP1-34 + CCGGGAUGCCUGGUACUGUU 20 420 TP53BP1-35 − CCGUGGAUGAUAGGUAGCUG 20 421 TP53BP1-36 + CGACCUAGGGAUCGAUCUGG 20 422 TP53BP1-37 + CGCAGCUACCUAUCAUCCAC 20 423 TP53BP1-38 + CGGCGACCUAGGGAUCGAUC 20 424 TP53BP1-39 + UCCGAUAUUGUGUGACGUGA 20 425 TP53BP1-40 + UGGCGGGCGGCGGCAGCGAA 20 426

Table II.2A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., TP53BP1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the TP53BP1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the TP53BP1 gene.

TABLE II.2A Exemplary HDR-enhancing gRNAs Targeting a TP53B1 Gene A high level of orthogonality, starts with a G, PAM is NNGRRT 2A S. aureus Target 1st Tier DNA Site Seq gRNA Name Strand Targeting Domain Length ID  TP53BP1- + GAGUGCAGUGGGCUCUGAAGGC 22 427 41 TP53BP1- + GCGGUGGCGACAGCGGCGACCU 22 428 42

Table II.2B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., TP53BP1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the TP53BP1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the TP53BP1 gene.

TABLE II.2B Exemplary HDR-enhancing gRNAs Targeting a TP53B1 Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 2B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID TP53BP1-43 − CGCCCGCCACUCAAGAAAUCCC 22 429 TP53BP1-44 − CGUGGAUGAUAGGUAGCUGCGG 22 430 TP53BP1-45 + CUGGUACUGUUUGGAGAGAAAU 22 431 TP53BP1-46 + UACCUAUCAUCCACGGGAUUUC 22 432 TP53BP1-47 + UCGAGCGCGAGGGACCUCCCGC 22 433 TP53BP1-48 + UCUUGAGUGGCGGGCGGCGGCA 22 434 TP53BP1-49 + UGCCGCAGCUACCUAUCAUCCA 22 435 TP53BP1-50 + UUGUGUGACGUGACGGGAAAGG 22 436 TP53BP1-51 − UUUCCCGUCACGUCACACAAUA 22 437

Table II.3A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RIF1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RIF1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RIF1 gene.

TABLE II.3A Exemplary HDR-enhancing gRNAs Targeting a RIF1 Gene A high level of orthogonality, and starts with a G 3A S. pyogenes Target 1st Tier DNA Site Seq gRNA Name Strand Targeting Domain Length ID RIF1-1 − GAACGAGGCAUCUCGCCGCG 20 438 RIF1-2 − GAGCUCGACUUUCCCAGCUC 20 439 RIF1-3 + GAUAAAUAUCGGGGUGACAG 20 440 RIF1-4 − GCCCAGGAGUGCGCGGGAGU 20 441 RIF1-5 + GCCGCCAUCUUGGUCUAGGA 20 442 RIF1-6 − GCGCGGGAGUAGGUUAGGCC 20 443 RIF1-7 − GGAGGAGAUCGGCGGAGGGC 20 444 RIF1-8 − GGAGUGCGCGGGAGUAGGUU 20 445 RIF1-9 − GGCAGACUGAGGGUUCCCCG 20 446 RIF1-10 − GGCAUCUCGCCGCGAGGGGG 20 447 RIF1-11 + GGCCCGCCCAGCCGCCAUCU 20 448 RIF1-12 + GGCGAGAUGCCUCGUUCCCC 20 449 RIF1-13 − GGGAGGAGAUCGGCGGAGGG 20 450 RIF1-14 − GGGAGUAGGUUAGGCCUGGC 20 451 RIF1-15 − GGGCAGCUUUCAACAGAGGG 20 452 RIF1-16 + GGGGUGACAGUGGUAGGCCG 20 453 RIF1-17 + GGGUGACAGUGGUAGGCCGC 20 454 RIF1-18 + GGUGACAGUGGUAGGCCGCG 20 455 RIF1-19 + GUCGAGCUCUGGCAGCGUCU 20 456 RIF1-20 + GUGAGUAAACAGCCGGAGCU 20 457

Table II.31B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RIF1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RIF1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RIF1 gene.

TABLE II.3B Exemplary HDR-enhancing gRNAs Targeting a RIF1 Gene A high level of orthogonality 3B S. pyogenes Target 2nd Tier DNA Site Seq gRNA Name Strand Targeting Domain Length ID RIF1-21 + AACCUACUCCCGCGCACUCC 20 458 RIF1-22 − AACGAGGCAUCUCGCCGCGA 20 459 RIF1-23 + AAUAUCGGGGUGACAGUGGU 20 460 RIF1-24 − ACGAGGCAUCUCGCCGCGAG 20 461 RIF1-25 + AGGGAGGCGAUCGAUAACUC 20 462 RIF1-26 + AGUCGAGCUCUGGCAGCGUC 20 463 RIF1-27 − CCACCUAGGAAGAUCAGGAC 20 464 RIF1-28 − CCCCGAUAUUUAUCCCACCU 20 465 RIF1-29 − CCUAGACCAAGAUGGCGGCU 20 466 RIF1-30 − CGAGGCAUCUCGCCGCGAGG 20 467 RIF1-31 + CGCACGCGUGAGUAAACAGC 20 468 RIF1-32 + CGGCGAGAUGCCUCGUUCCC 20 469 RIF1-33 + CGUGAGUAAACAGCCGGAGC 20 470 RIF1-34 − UCCAACAGUCAGCGGCACAC 20 471 RIF1-35 + UCCGGUGUGCCGCUGACUGU 20 472 RIF1-36 − UCUCGCCGCGAGGGGGCGGA 20 473 RIF1-37 + UGGCUCGAACUUCUCCCGCC 20 474 RIF1-38 + UGUGCCGCUGACUGUUGGAU 20 475 RIF1-39 − UUAUCCCACCUAGGAAGAUC 20 476 RIF1-40 + UUCCUAGGUGGGAUAAAUAU 20 477

Table II.4A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RIF1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RIF1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RIF1 gene.

TABLE II.4A Exemplary HDR-enhancing gRNAs Targeting a RIF1 Gene A high level of orthogonality, starts with a G, PAM is NNGRRT 4A S. aureus Target 1st Tier DNA Site Seq gRNA Name Strand Targeting Domain Length ID RIF1-41 + GAAAGUCGAGCUCUGGCAGCGU 22 478 RIF1-42 − GGCAUCUCGCCGCGAGGGGGCG 22 479 RIF1-43 − GGGGGCGGAGGGUGGGCAGACU 22 480

Table II.41B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RIF1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RIF1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RIF1 gene.

TABLE II.4B Exemplary HDR-enhancing gRNAs Targeting a RIF1 Gene A high level of orthogonality, and PAM is NNGRRT S. aureus 4B 2nd Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID RIF1-44 + AACUCCGGUGUGCCGCUGACUG 22 481 RIF1-45 − AAGAUGGCGGCUGGGCGGGCCC 22 482 RIF1-46 − AAUCCAACAGUCAGCGGCACAC 22 483 RIF1-47 + ACCCUGUCCUGAUCUUCCUAGG 22 484 RIF1-48 − AUCCCACCUAGGAAGAUCAGGA 22 485 RIF1-49 − CUGGGCGGGCCCAGGAGUGCGC 22 486 RIF1-50 + UAGGAGGGAGCGCGCCGCACGC 22 487 RIF1-51 + UCUUCCUAGGUGGGAUAAAUAU 22 488

Table II.5A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., PAXIP1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the PAXIP1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the PAXIP1 gene.

TABLE II.5A Exemplary HDR-enhancing gRNAs Targeting a PAXIP1 Gene A high level of orthogonality, and starts with a G 5A S. pyogenes Target 1st Tier DNA Site Seq gRNA Name Strand Targeting Domain Length ID PAXIP1-1 − GAACAUCUCCUCAGGAACUU 20 489 PAXIP1-2 − GCCCCCACUCGCCCCGCCAA 20 490 PAXIP1-3 + GCCGUUGGCGGGGCGAGUGG 20 491 PAXIP1-4 − GCGCCGAGCGCCCGAAGCGC 20 492 PAXIP1-5 + GCGCCGCCGCGGAGCCUCCC 20 493 PAXIP1-6 + GCGCGCGGCUCCCGCGCUUC 20 494 PAXIP1-7 + GCGCGGGCAGGGCCGUUGGC 20 495 PAXIP1-8 + GCGCUCCCCCUCGGUGGCCG 20 496 PAXIP1-9 + GCGGGAUGGUGCGUCCCGCA 20 497 PAXIP1-10 + GCUCCCGCGCUUCGGGCGCU 20 498 PAXIP1-11 + GGACCCCGAUUCGCAGGACC 20 499 PAXIP1-12 + GGACCGGGCCCGGGCUGCGC 20 500 PAXIP1-13 + GGCCGUUGGCGGGGCGAGUG 20 501 PAXIP1-14 + GGCGCUCCCCCUCGGUGGCC 20 502 PAXIP1-15 + GGCGGGAUGGUGCGUCCCGC 20 503 PAXIP1-16 + GGCUGCGCGGGCAGGGCCGU 20 504 PAXIP1-17 − GGGAGCCGCGCGCGCCCUGC 20 505 PAXIP1-18 + GGGAGCGGACCCCGAUUCGC 20 506 PAXIP1-19 − GGGCCCGGUCCUGCGAAUCG 20 507 PAXIP1-20 + GGGCCGUUGGCGGGGCGAGU 20 508

Table II.51 provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., PAXIP1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the PAXIP1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the PAXIP1 gene.

TABLE II.5B Exemplary HDR-enhancing gRNAs Targeting a PAXIP1 Gene A high level of orthogonality 5B S. pyogenes Target 2nd Tier DNA Site Seq gRNA Name Strand Targeting Domain Length ID PAXIP1-21 − AACAUCUCCUCAGGAACUUU 20 509 PAXIP1-22 − CAUGAUCGCGGCGGCCCGGG 20 510 PAXIP1-23 + CCAGGCGCCCAAAGUUCCUG 20 511 PAXIP1-24 − CCGACAUGAUCGCGGCGGCC 20 512 PAXIP1-25 + CCGAUUCGCAGGACCGGGCC 20 513 PAXIP1-26 − CCGGGCCCGGUCCUGCGAAU 20 514 PAXIP1-27 + CCGGGCCGCCGCGAUCAUGU 20 515 PAXIP1-28 − CCUCAGGAACUUUGGGCGCC 20 516 PAXIP1-29 − CGACAUGAUCGCGGCGGCCC 20 517 PAXIP1-30 + CGAUUCGCAGGACCGGGCCC 20 518 PAXIP1-31 + CGCCGCGAUCAUGUCGGACC 20 519 PAXIP1-32 − CGCCUGGUCCGACAUGAUCG 20 520 PAXIP1-33 − CGCGCCGAGCGCCCGAAGCG 20 521 PAXIP1-34 + CGCGCGCGGCUCCCGCGCUU 20 522 PAXIP1-35 + CGCGCUUCGGGCGCUCGGCG 20 523 PAXIP1-36 + CGGACCCCGAUUCGCAGGAC 20 524 PAXIP1-37 − CGGGACGCACCAUCCCGCCC 20 525 PAXIP1-38 − CGGGCCCGGUCCUGCGAAUC 20 526 PAXIP1-39 − CUGGUCCGACAUGAUCGCGG 20 527 PAXIP1-40 + UGCGUCCCGCAGGGCGCGCG 20 528

Table II.6A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., PAXIP1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the PAXIP1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the PAXIP1 gene.

TABLE II.6A Exemplary HDR-enhancing gRNAs Targeting a PAXIP1 Gene A high level of orthogonality, starts with a G, PAM is NNGRRT 6A S. aureus Target 1st Tier DNA Site Seq gRNA Name Strand Targeting Domain Length ID PAXIP1-41 − GCCCGGGCCCGGUCCUGCGAAU 22 529 PAXIP1-42 + GGCGCCGCGGGGGCCGGGGGCG 22 530

Table II.6B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., PAXIP1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the PAXIP1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the PAXIP1 gene.

TABLE II.6B Exemplary HDR-enhancing gRNAs Targeting a PAXIP1 Gene A high level of orthogonality, and PAM is NNGRRT 6B S. aureus Target 2nd Tier DNA Site Seq gRNA Name Strand Targeting Domain Length ID PAXIP1-43 − CGCGCAGCCCGGGCCCGGUCCU 22 531 PAXIP1-44 + CGCGGGCAGGGCCGUUGGCGGG 22 532 PAXIP1-45 + CGCUCCCCCUCGGUGGCCGGGG 22 533

Table II.7A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., XRCC6 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the XRCC6 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the XRCC6 gene.

TABLE II.7A Exemplary HDR-enhancing gRNAs Targeting a XRCC6 Gene A high level of orthogonality, and starts with a G 7A S. pyogenes Target 1st Tier DNA Site Seq gRNA Name Strand Targeting Domain Length ID XRCC6-1 − GACGACAAUCCACGCAUGCG 20 534 XRCC6-2 − GAGCGAAGCGGGACGAGGCC 20 535 XRCC6-3 + GAGGCGGCACCUCGCGUUUG 20 536 XRCC6-4 + GAGGGCCCACACGGAAGAGG 20 537 XRCC6-5 − GAGGUGCCGCCUCCUUCCCG 20 538 XRCC6-6 − GAUAACGGCCCGCUUACCUU 20 539 XRCC6-7 + GCACAUGCGUGAUGACGUAG 20 540 XRCC6-8 − GCAUGCGCGGGCCCUGUACU 20 541 XRCC6-9 + GCCCCCAUAGCCUUGCUAGA 20 542 XRCC6-10 − GCCCCGCCCCUUCCUACGUC 20 543 XRCC6-11 − GCCCGCUUACCUUUGGCGCA 20 544 XRCC6-12 + GCCUUAAGUGUGCGAAUCCG 20 545 XRCC6-13 − GCGAGACCGACCGAGCGAAG 20 546 XRCC6-14 + GGACAUAGGUAGAAGCUGGU 20 547 XRCC6-15 − GGCCCGCUUACCUUUGGCGC 20 548 XRCC6-16 + GGGCGGGGCUUUGCCGAAGG 20 549 XRCC6-17 + GGGGCGGGGCUCUCGCUGAU 20 550 XRCC6-18 + GGGGCGGGGCUUUGCCGAAG 20 551 XRCC6-19 + GUACAGGGCCCGCGCAUGCG 20 552 XRCC6-20 + GUUGAUUGGGACCGAGUACA 20 553

Table II.71 provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., XRCC6 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the XRCC6 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the XRCC6 gene.

TABLE II.7B Exemplary HDR-enhancing gRNAs Targeting a XRCC6 Gene A high level of orthogonality 7B S. pyogenes Target 2nd Tier DNA Site Seq gRNA Name Strand Targeting Domain Length ID XRCC6-21 + ACCACGCUCCUUCCUCGGGA 20 554 XRCC6-22 − ACGACAAUCCACGCAUGCGC 20 555 XRCC6-23 + AUGACGUAGAGGGCGUUGAU 20 556 XRCC6-24 + CACAUGCGUGAUGACGUAGA 20 557 XRCC6-25 − CGACCGAGCGAAGCGGGACG 20 558 XRCC6-26 − CGAGACCGACCGAGCGAAGC 20 559 XRCC6-27 − CGCACUAUAUCGCGUCAGGC 20 560 XRCC6-28 + CGCCCCCAUAGCCUUGCUAG 20 561 XRCC6-29 + CGGGGCUCUCGCUGAUGGGU 20 562 XRCC6-30 + CGUUGAUUGGGACCGAGUAC 20 563 XRCC6-31 − CUAACGCUAACCCUCUAGCA 20 564 XRCC6-32 + CUCGUCCCGCUUCGCUCGGU 20 565 XRCC6-33 + CUGAUGGGUUGGCUUUCGUC 20 566 XRCC6-34 + UCCCUGCGCCAAAGGUAAGC 20 567 XRCC6-35 − UCCUCGGAUUCGCACACUUA 20 568 XRCC6-36 − UCGAGUCUGUCGCUGCUCCU 20 569 XRCC6-37 + UGACGUAGAGGGCGUUGAUU 20 570 XRCC6-38 + UGGUCGCUUCCCUGCGCCAA 20 571 XRCC6-39 − UGUGCGCACUAUAUCGCGUC 20 572 XRCC6-40 + UGUUGUUCGCCAGCUAGGCC 20 573

Table II.8A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., XRCC6 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the XRCC6 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the XRCC6 gene.

TABLE II.8A Exemplary HDR-enhancing gRNAs Targeting a XRCC6 Gene A high level of orthogonality, starts with a G, PAM is NNGRRT 8A S. aureus Target 1st Tier DNA Site Seq gRNA Name Strand Targeting Domain Length ID XRCC6-41 + GAAGGGGGCGGGGCUCUCGCUG 22 574 XRCC6-42 − GCCUAGCUGGCGAACAACACAA 22 575 XRCC6-43 + GCGCGCCCCCAUAGCCUUGCUA 22 576 XRCC6-44 + GGACAUAGGUAGAAGCUGGUUG 22 577 XRCC6-45 + GGUUAGCGUUAGCCUUAAGUGU 22 578 XRCC6-46 − GUCUCGAGUCUGUCGCUGCUCC 22 579

Table II.8B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., XRCC6 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the XRCC6 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the XRCC6 gene.

TABLE II.8B Exemplary HDR-enhancing gRNAs Targeting a XRCC6 Gene A high level of orthogonality, and PAM is NNGRRT 8B S. aureus Target 2nd Tier DNA Site Seq gRNA Name Strand Targeting Domain Length ID XRCC6-47 + ACACGGAAGAGGGGGCGGGGGC 22 580 XRCC6-48 + ACGUAGAGGGCGUUGAUUGGGA 22 581 XRCC6-49 + CGAGUACAGGGCCCGCGCAUGC 22 582 XRCC6-50 − UUCCCGAGGAAGGAGCGUGGUC 22 583

Table II.9A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., XRCC5 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the XRCC5 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the XRCC5 gene.

TABLE II.9A Exemplary HDR-enhancing gRNAs Targeting a XRCC5 Gene A high level of orthogonality, and starts with a G 9A S. pyogenes Target 1st Tier DNA Site Seq gRNA Name Strand Targeting Domain Length ID XRCC5-1 + GAAGCGAGUUGCGACACGGC 20 584 XRCC5-2 + GACCGGCAACAUGGUGCGGU 20 585 XRCC5-3 + GACUUGGGCUUUACCCGGAC 20 586 XRCC5-4 + GAGAAUGUGCGCAUGCUCGG 20 587 XRCC5-5 − GAGCCGCUUCGUUUCCUGCU 20 588 XRCC5-6 − GCACCAUGUUGCCGGUCCUC 20 589 XRCC5-7 − GCCGUGUCGCAACUCGCUUC 20 590 XRCC5-8 + GCGCCUGAGGACCGGCAACA 20 591 XRCC5-9 − GCGCUUUGGUCGCUUCUUCC 20 592 XRCC5-10 + GCUAUCUGCCGCUUGUCCAC 20 593 XRCC5-11 + GGAAUCUGCGCGAGCUCGGC 20 594 XRCC5-12 + GGAGAGAAUGUGCGCAUGCU 20 595 XRCC5-13 + GGCCGGAAUCUGCGCGAGCU 20 596 XRCC5-14 − GGCGCUUUGGUCGCUUCUUC 20 597 XRCC5-15 + GGGAAUCUGCGCAAGCUCGG 20 598 XRCC5-16 + GGGAAUCUGCGCAUGCUCGG 20 599 XRCC5-17 − GGGGCGGGGAAACCGUGCCC 20 600 XRCC5-18 − GGUGGACAAGCGGCAGAUAG 20 601 XRCC5-19 − GUGUCGCAACUCGCUUCCGG 20 602 XRCC5-20 − GUUUCCUGCUAGGCCUGAAA 20 603

Table II.91B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., XRCC5 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the XRCC5 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the XRCC5 gene.

TABLE II.9B Exemplary HDR-enhancing gRNAs Targeting a XRCC5 Gene A high level of orthogonality 9B S. pyogenes Target 2nd Tier DNA Site Seq gRNA Name Strand Targeting Domain Length ID XRCC5-21 + ACCGGAAGCGAGUUGCGACA 20 604 XRCC5-22 + ACCGGCAACAUGGUGCGGUC 20 605 XRCC5-23 + ACGGUUUCCCCGCCCCUUUC 20 606 XRCC5-24 − ACUCGCUUCCGGUGGACAAG 20 607 XRCC5-25 + ACUUGGGCUUUACCCGGACU 20 608 XRCC5-26 + AGAAGCGACCAAAGCGCCUG 20 609 XRCC5-27 + CAUGGUGCGGUCGGGGAAUA 20 610 XRCC5-28 + CCACACGCUCCCGACUACGG 20 611 XRCC5-29 + CCGCCCCUUUCAGGCCUAGC 20 612 XRCC5-30 − CCGCCGUAGUCGGGAGCGUG 20 613 XRCC5-31 + CCGGCAACAUGGUGCGGUCG 20 614 XRCC5-32 − CGCCGAGCUCGCGCAGAUUC 20 615 XRCC5-33 + CGCUCCCGACUACGGCGGAA 20 616 XRCC5-34 − CGUUUCCUGCUAGGCCUGAA 20 617 XRCC5-35 − CUCUCCAUUCCGCCGUAGUC 20 618 XRCC5-36 + CUGCGCAUGCUCAGAGUUCC 20 619 XRCC5-37 − UCUCUCCAUUCCGCCGUAGU 20 620 XRCC5-38 + UGCGCAUGCUCAGAGUUCCG 20 621 XRCC5-39 − UUGCCGGUCCUCAGGCGCUU 20 622 XRCC5-40 − UUUGGUCGCUUCUUCCGGGC 20 623

Table II.10A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., XRCC5 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the XRCC5 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the XRCC5 gene.

TABLE II.10A Exemplary HDR-enhancing gRNA Targeting a XRCC5 Gene A high level of orthogonality, starts with a G, PAM is NNGRRT 10A S. aureus Target 1st Tier DNA Site Seq gRNA Name Strand Targeting Domain Length ID XRCC5-41 + GACCGGCAACAUGGUGCGGUCG 22 624

Table II.10B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., XRCC5 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the XRCC5 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the XRCC5 gene.

TABLE II.10B Exemplary HDR-enhancing gRNAs Targeting a XRCC5 Gene A high level of orthogonality, and PAM is NNGRRT 10B S. aureus Target 2nd Tier DNA Site Seq gRNA Name Strand Targeting Domain Length ID XRCC5-42 + AGAGAAUCUGCGCAUGCUCGGC 22 625 XRCC5-43 + AGAGAAUGUGCGCAUGCUCGGC 22 626 XRCC5-44 + AUCUGCCGCUUGUCCACCGGAA 22 627 XRCC5-45 + CACCACACGCUCCCGACUACGG 22 628 XRCC5-46 + CCGGAAUCUGCGCGAGCUCGGC 22 629 XRCC5-47 + CGGGAAUCUGCGCAAGCUCGGC 22 630 XRCC5-48 + CGGGAAUCUGCGCAUGCUCGGA 22 631 XRCC5-49 + CGGGAAUCUGCGCAUGCUCGGC 22 632 XRCC5-50 + CUCCCGACUACGGCGGAAUGGA 22 633 XRCC5-51 + UCGGCGGGAAUCUGCGCAUGCU 22 634

Table II.11A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., PRKDC gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the PRKDC gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the PRKDC gene.

TABLE II.11A Exemplary HDR-enhancing gRNAs Targeting a PRKDC Gene A high level of orthogonality, and starts with a G 11A S. pyogenes Target 1st Tier DNA Site Seq gRNA Name Strand Targeting Domain Length ID PRKDC-1 + GCACGCGCGGGAGCGGGACU 20 635 PRKDC-2 + GCAGCCCCGCCUCCGCGCGU 20 636 PRKDC-3 + GCCUUCCCGCAGGGGUCCCC 20 637 PRKDC-4 − GCGCCCGCUCGGCCCGGACC 20 638 PRKDC-5 − GCGCGUGCGCCCGCUCGGCC 20 639 PRKDC-6 − GCGGCGGCAGGAACUUUCCC 20 640 PRKDC-7 + GCGGGACUCGGCGGCAUGGC 20 641 PRKDC-8 + GCGGGCGCACGCGCGGGAGC 20 642 PRKDC-9 − GGAAAUGCCCCUACGCGCGG 20 643 PRKDC-10 − GGAGCAACGCACACCGGCUC 20 644 PRKDC-11 + GGCAUGGCGGGCUCCGGAGC 20 645 PRKDC-12 + GGCCAAAGAGGCGCGCUUAC 20 646 PRKDC-13 − GGCCAGUAAGCGCGCCUCUU 20 647 PRKDC-14 + GGCCGAGCGGGCGCACGCGC 20 648 PRKDC-15 + GGCCUUCCCGCAGGGGUCCC 20 649 PRKDC-16 − GGGACCCCUGCGGGAAGGCC 20 650 PRKDC-17 + GGGAGCGGGACUCGGCGGCA 20 651 PRKDC-18 + GGGCCGAGCGGGCGCACGCG 20 652 PRKDC-19 + GUAGGGGCAUUUCCGGGUCC 20 653 PRKDC-20 + GUGUGCGUUGCUCCCUGCUG 20 654

Table II.11B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., PRKDC gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the PRKDC gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the PRKDC gene.

TABLE II.11B Exemplary HDR-enhancing gRNAs Targeting a PRKDC Gene A high level of orthogonality 11B S. pyogenes Target 2nd Tier DNA Site Seq gRNA Name Strand Targeting Domain Length ID PRKDC-21 + AAGAGGCGCGCUUACUGGCC 20 655 PRKDC-22 − AAUGCCCCUACGCGCGGAGG 20 656 PRKDC-23 − ACUUUCCCGGGGACCCCUGC 20 657 PRKDC-24 + AGCCCCGCCUCCGCGCGUAG 20 658 PRKDC-25 + AGCGGGACUCGGCGGCAUGG 20 659 PRKDC-26 − AUGCCCCUACGCGCGGAGGC 20 660 PRKDC-27 + AUUUCCGGGUCCGGGCCGAG 20 661 PRKDC-28 + CAGCCCCGCCUCCGCGCGUA 20 662 PRKDC-29 + CAUGUUGAUUCGGGCCAAAG 20 663 PRKDC-30 − CCCGGAAAUGCCCCUACGCG 20 664 PRKDC-31 + CCGCGCGUAGGGGCAUUUCC 20 665 PRKDC-32 − CGAAUCAACAUGGAAACCUA 20 666 PRKDC-33 − CGCGGCGGCAGGAACUUUCC 20 667 PRKDC-34 − CGGCGGCAGGAACUUUCCCG 20 668 PRKDC-35 + CGUAGGGGCAUUUCCGGGUC 20 669 PRKDC-36 + CUCGGCGGCAUGGCGGGCUC 20 670 PRKDC-37 − CUCUUUGGCCCGAAUCAACA 20 671 PRKDC-38 + UCCGCGCGUAGGGGCAUUUC 20 672 PRKDC-39 − UGCCCCUACGCGCGGAGGCG 20 673 PRKDC-40 + UUUCCGGGUCCGGGCCGAGC 20 674

Table II.12A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., PRKDC gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the PRKDC gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the PRKDC gene.

TABLE II.12A Exemplary HDR-enhancing gRNAs Targeting a PRKDC Gene A high level of orthogonality, starts with a G, PAM is NNGRRT 12A S. aureus Target 1st Tier DNA Site Seq gRNA Name Strand Targeting Domain Length ID PRKDC-41 + GCCUCCGCGCGUAGGGGCAUUU 22 675 PRKDC-42 − GGCUCCGGAGCCCGCCAUGCCG 22 676

Table II.12B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., PRKDC gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the PRKDC gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the PRKDC gene.

TABLE II.12B Exemplary HDR-enhancing gRNAs Targeting a PRKDC Gene A high level of orthogonality, and PAM is NNGRRT 12B S. aureus Target 2nd Tier DNA Site Seq gRNA Name Strand Targeting Domain Length ID PRKDC-43 − CCAGUAAGCGCGCCUCUUUGGC 22 677 PRKDC-44 + CUUACUGGCCAGGCCUUCCCGC 22 678

Table II.13A provides exemplary targeting domains of a gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., LIG4 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the LIG4 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the LIG4 gene.

TABLE II.13A Exemplary HDR-enhancing gRNAs Targeting a LIG4 Gene A high level of orthogonality, and starts with a G 13A S. pyogenes Target 1st Tier DNA Site Seq gRNA Name Strand Targeting Domain Length ID LIG4-1 + GACGUCAGGUGGGAAGGGUG 20 679 LIG4-2 − GCAGCCAGGCUCGCGAUGGG 20 680 LIG4-3 − GCAGGCGCAGGGGAGACCCG 20 681 LIG4-4 − GCCAGGCUCGCGAUGGGAGG 20 682 LIG4-5 + GCCCGGUGACUGCAAGGCCC 20 683 LIG4-6 − GCGCAGGCGCAGGGGAGACC 20 684 LIG4-7 + GCGCCUGCGCGGCGAGCAGC 20 685 LIG4-8 − GCUCGCGAUGGGAGGUGGGG 20 686 LIG4-9 − GCUGCUCGCCGCGCAGGCGC 20 687 LIG4-10 + GCUUCAGGCUUGACGUCAGG 20 688 LIG4-11 + GCUUGAGCCCGGUGACUGCA 20 689 LIG4-12 − GGCGCAGCCAGGCUCGCGAU 20 690 LIG4-13 + GGCGCCAGCUUCCGGCUUAG 20 691 LIG4-14 − GGCUCGCGAUGGGAGGUGGG 20 692 LIG4-15 + GGCUUGACGUCAGGUGGGAA 20 693 LIG4-16 + GGGGCGGUUGGGAGGUUGGG 20 694 LIG4-17 + GGGUCUCCCCUGCGCCUGCG 20 695 LIG4-18 + GGUGGCAGGUGGGGGCGGUU 20 696 LIG4-19 − GUCACCGGGCUCAAGCACGC 20 697 LIG4-20 + GUGGGGGCGGUUGGGAGGUU 20 698

Table II.13B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., LIG4 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the LIG4 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the LIG4 gene.

TABLE II.13B Exemplary HDR-enhancing gRNAs Targeting a LIG4 Gene A high level of orthogonality 13B S. pyogenes Target 2nd Tier DNA Targeting Site Seq gRNA Name Strand Domain Length ID LIG4-21 − ACCCGGGGCCUUGCAGUCAC 20 699 LIG4-22 + ACGUCAGGUGGGAAGGGUGU 20 700 LIG4-23 + AGGCUUGACGUCAGGUGGGA 20 701 LIG4-24 − CAGGCUCGCGAUGGGAGGUG 20 702 LIG4-25 + CAUCGCGAGCCUGGCUGCGC 20 703 LIG4-26 + CAUCUUCUGGCGCCAGCUUC 20 704 LIG4-27 − CCAGGCUCGCGAUGGGAGGU 20 705 LIG4-28 + CCCACCUCCCAUCGCGAGCC 20 706 LIG4-29 − CCCGGGGCCUUGCAGUCACC 20 707 LIG4-30 + CCCGGUGACUGCAAGGCCCC 20 708 LIG4-31 − CGCAGGCGCAGGGGAGACCC 20 709 LIG4-32 + CGGCGAGCAGCUGGCGGAAC 20 710 LIG4-33 − CGGCGCAGCCAGGCUCGCGA 20 711 LIG4-34 + CGGCUUAGCGGCUGAGCUUC 20 712 LIG4-35 − CUGAAGCUCAGCCGCUAAGC 20 713 LIG4-36 + CUGGCGGAACCGGCAUCUUC 20 714 LIG4-37 − UCAAGCACGCCGGCGCAGCC 20 715 LIG4-38 − UCAGCCGCUAAGCCGGAAGC 20 716 LIG4-39 + UGAGCUUCAGGCUUGACGUC 20 717 LIG4-40 + UGCGCCGGCGUGCUUGAGCC 20 718

Table II.14A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., LIG4 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the LIG4 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the LIG4 gene.

TABLE II.14A Exemplary HDR-enhancing gRNA Targeting a LIG4 Gene A high level of orthogonality, starts with a G, PAM is NNGRRT 14A S. aureus Target 1st Tier DNA Targeting Site Seq gRNA Name Strand Domain Length ID LIG4-41 + GGUUGGGGGGGGUUGGGGUGGG 22 719

Table II.14B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., LIG4 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the LIG4 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the LIG4 gene.

TABLE II.14B Exemplary HDR-enhancing gRNAs Targeting a LIG4 Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 14B gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID LIG4-42 + CGGUUGGGAGGUUGGGGGGGGU 22 720 LIG4-43 + UGAGCCCGGUGACUGCAAGGCC 22 721 LIG4-44 + UGGGGGCGGUUGGGAGGUUGGG 22 722 LIG4-45 + UUCAGGCUUGACGUCAGGUGGG 22 723 LIG4-46 + UUGACGUCAGGUGGGAAGGGUG 22 724

Table II.15A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., XRCC4 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the XRCC4 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the XRCC4 gene.

TABLE II.15A Exemplary HDR-enhancing gRNAs Targeting a XRCC4 Gene S. pyogenes A high level of orthogonality, and starts with a G 1st Tier 15A gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID XRCC4-1 + GAAGUAGCUGAUACUCUCAU 20 725 XRCC4-2 − GACAAGCCCAACCGGACGGC 20 726 XRCC4-3 − GACGGCUGGAGAGGGCGAGA 20 727 XRCC4-4 − GAGAGGGCGAGAAGGGCAGA 20 728 XRCC4-5 + GAGAGGUAGGAUCCGGAAGU 20 729 XRCC4-6 + GAUCACGUCCCGCAGGCCGG 20 730 XRCC4-7 − GAUCUAAAUCCCGCCUUUUC 20 731 XRCC4-8 + GCACCGCCUACCAAGACGGG 20 732 XRCC4-9 − GCCCAACCGGACGGCUGGAG 20 733 XRCC4-10 + GCCCUCUCCAGCCGUCCGGU 20 734 XRCC4-11 + GCGGGCGUUUUGGAAGAUAC 20 735 XRCC4-12 + GGAGAGGUAGGAUCCGGAAG 20 736 XRCC4-13 + GGAUUUAGAUCACGUCCCGC 20 737 XRCC4-14 + GGCGGUUAAGACACUAGGAU 20 738 XRCC4-15 − GGUGCCGUGACAAGCCCAAC 20 739

Table II.15B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., XRCC4 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the XRCC4 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the XRCC4 gene.

TABLE II.15B Exemplary HDR-enhancing gRNAs Targeting a XRCC4 Gene S. pyogenes A high level of orthogonality 2nd Tier 15B gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID XRCC4-16 − ACGCCCGCUUUCACAGAUCA 20 740 XRCC4-17 + ACGGCACCGCCUACCAAGAC 20 741 XRCC4-18 + AGACGGGCGGUUAAGACACU 20 742 XRCC4-19 − AGAGUAUCAGCUACUUCCGC 20 743 XRCC4-20 + AGAUACCGGAAGUAGAGUCA 20 744 XRCC4-21 − AGCUACUUCCGCCGGCCUGC 20 745 XRCC4-22 + AGUCACGGAGAGGUAGGAUC 20 746 XRCC4-23 − AGUGUCUUAACCGCCCGUCU 20 747 XRCC4-24 + AUCUGUGAAAGCGGGCGUUU 20 748 XRCC4-25 + CACGGCACCGCCUACCAAGA 20 749 XRCC4-26 − CCCAACCGGACGGCUGGAGA 20 750 XRCC4-27 + CCCUCUCCAGCCGUCCGGUU 20 751 XRCC4-28 + CCGGAAGUAGAGUCACGGAG 20 752 XRCC4-29 + CCGUCCGGUUGGGCUUGUCA 20 753 XRCC4-30 − CCGUGACAAGCCCAACCGGA 20 754 XRCC4-31 − CUAAAUCCCGCCUUUUCCGG 20 755 XRCC4-32 − UAACCGCCCGUCUUGGUAGG 20 756 XRCC4-33 − UCCCGCCUUUUCCGGUGGAG 20 757 XRCC4-34 − UCUUAACCGCCCGUCUUGGU 20 758 XRCC4-35 + UUAGAUCACGUCCCGCAGGC 20 759

Table II.16A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., XRCC4 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the XRCC4 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the XRCC4 gene.

TABLE II.16A Exemplary HDR-enhancing gRNAs Targeting a XRCC4 Gene S. aureus A high level of orthogonality, starts with a G, PAM is NNGRRT 1st Tier 16A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID XRCC4-36 + GCGUUUUGGAAGAUACCGGAAG 22 760 XRCC4-37 + GGCUCCUCUCCACCGGAAAAGG 22 761

Table II.16B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., XRCC4 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the XRCC4 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the XRCC4 gene.

TABLE II.16B Exemplary HDR-enhancing gRNAs Targeting a XRCC4 Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 16B gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID XRCC4-38 − AGAUCAAGGUUUUGCAACCAAU 22 762 XRCC4-39 − AUUUAAAGAGGCAGCCCCACUU 22 763 XRCC4-40 + CCAAGACGGGCGGUUAAGACAC 22 764 XRCC4-41 + CGGAAGUAGAGUCACGGAGAGG 22 765

Table II.17A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., NHEJ1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the NHEJ1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the NHEJ1 gene.

TABLE II.17A Exemplary HDR-enhancing gRNAs Targeting a NHEJ1 Gene S. pyogenes A high level of orthogonality, and starts with a G 1st Tier 17A gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID NHEJ1-1 + GCAGUCCGCUGGCUGCUGCC 20 766 NHEJ1-2 − GCCAGCGGACUGCGCACGCG 20 767 NHEJ1-3 − GCCCGCUCGCGCAAACCGAA 20 768 NHEJ1-4 + GCCUUUCGGUUUGCGCGAGC 20 769 NHEJ1-5 + GCGACGAAGCCGCUGGUGGC 20 770 NHEJ1-6 − GCGAUUCCACCUACCGUCAG 20 771 NHEJ1-7 − GCGCUCCCUCCAGGGAGAAA 20 772 NHEJ1-8 − GCGGCUUCGUCGCACCAAAC 20 773 NHEJ1-9 − GCGUCUGAGCAGCCCCUCGC 20 774 NHEJ1-10 + GCUCGAGUGAAGGUACUCGU 20 775 NHEJ1-11 + GCUGCCCGGCGUGGAUGGUA 20 776 NHEJ1-12 + GCUGCUCAGACGCUGCGGGU 20 777 NHEJ1-13 − GGCCUAUGCCUGGCGUGGGC 20 778 NHEJ1-14 + GGCCUUUCGGUUUGCGCGAG 20 779 NHEJ1-15 + GGCGCUCUCGCGGCCGCUGA 20 780 NHEJ1-16 − GGUCUUGGGAUACAGGGGCG 20 781 NHEJ1-17 + GGUGGAAUCGCGUUCGAGUC 20 782 NHEJ1-18 + GUGCGUGCGGCUAAGAGAGU 20 783 NHEJ1-19 + GUGGAAUCGCGUUCGAGUCC 20 784 NHEJ1-20 + GUUUGGUGCGACGAAGCCGC 20 785

Table II.17B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., NHEJ1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the NHEJ1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site TS S) of the NHEJ1 gene.

TABLE II.17B Exemplary HDR-enhancing gRNAs Targeting a NHEJ1 Gene S. pyogenes A high level of orthogonality 2nd Tier 17B gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID NHEJ1-21 + AAUCGCGUUCGAGUCCGGGC 20 786 NHEJ1-22 + ACCCUGCCUCCUCUUGCGGU 20 787 NHEJ1-23 − ACCGAAAGGCCUAGAGUAAG 20 788 NHEJ1-24 + CAGACGCUGCGGGUUGGCCC 20 789 NHEJ1-25 − CGAGCCCUACCAUCCACGCC 20 790 NHEJ1-26 + CGCUGGCCUUUUCUCCCUGG 20 791 NHEJ1-27 − CGCUUUCCCCCCACCGCAAG 20 792 NHEJ1-28 + CGGGCAGGAAAGCGUGCGUG 20 793 NHEJ1-29 + CGUGCGUGCGGCUAAGAGAG 20 794 NHEJ1-30 + CUCCACUUACCCUGGCCACU 20 795 NHEJ1-31 + CUGCCUCCUCUUGCGGUGGG 20 796 NHEJ1-32 + CUGCGGGUUGGCCCUGGCGC 20 797 NHEJ1-33 + UAAGAGAGUGGGCGCUCUCG 20 798 NHEJ1-34 − UCGAGCCCUACCAUCCACGC 20 799 NHEJ1-35 + UCGCGGCCGCUGACGGUAGG 20 800 NHEJ1-36 + UGCUGCCCGGCGUGGAUGGU 20 801 NHEJ1-37 + UGGAGGGAGCGCGCGCUGCC 20 802 NHEJ1-38 + UGGUGCGACGAAGCCGCUGG 20 803 NHEJ1-39 + UUCGGUUUGCGCGAGCGGGC 20 804 NHEJ1-40 − UUUCCCCCCACCGCAAGAGG 20 805

Table II.18A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., NHEJ1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the NHEJ1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site TS S) of the NHEJ1 gene.

TABLE II.18A Exemplary HDR-enhancing gRNAs Targeting a NHEJ1 Gene S. aureus A high level of orthogonality, starts with a G, PAM is NNGRRT 1st Tier 18A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID NHEJ1-41 + GUCCAGGGCAGGCCUCCGGGGG 22 806

Table II.18B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., NHEJ1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the NHEJ1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the NHEJ1 gene.

TABLE II.18B Exemplary HDR-enhancing gRNAs Targeting a NHEJ1 Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 18B gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID NHEJ1-42 − ACCGCAAGAGGAGGCAGGGUCU 22 807 NHEJ1-43 + AGCGAGGGGCUGCUCAGACGCU 22 808 NHEJ1-44 + AGGAAAGCGUGCGUGCGGCUAA 22 809 NHEJ1-45 + AGUCCGCUGGCUGCUGCCCGGC 22 810 NHEJ1-46 − AUGAGAGGAGCGCCCCAGUGGC 22 811 NHEJ1-47 + CCCUGGAGGGAGCGCGCGCUGC 22 812 NHEJ1-48 − CGCUCGCGCAAACCGAAAGGCC 22 813 NHEJ1-49 − CGUCGCACCAAACAGGCGACCA 22 814 NHEJ1-50 + CUGACGGUAGGUGGAAUCGCGU 22 815 NHEJ1-51 + UCUCGCGGCCGCUGACGGUAGG 22 816 NHEJ1-52 + UGCCCGGCGUGGAUGGUAGGGC 22 817 NHEJ1-53 − UUCCCCCCACCGCAAGAGGAGG 22 818

Table II.19A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., DCLRE1C gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the DCLRE8C gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the DCLRE1C gene.

TABLE II.19A Exemplary HDR-enhancing gRNAs Targeting a DCLRE1C Gene S. pyogenes A high level of orthogonality, and starts with a G 1st Tier 19A gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID DCLRE1C-1 + GAGUUCUUUCGAGGGGCAGA 20 819 DCLRE1C-2 − GCCGCGCGCUGCCUCGCCAU 20 820 DCLRE1C-3 − GCGCCGCCGAUCCCAGAGUC 20 821 DCLRE1C-4 + GCGCGGCUUCCCGGAAGUGG 20 822 DCLRE1C-5 + GCGCUAUGAGUUCUUUCGAG 20 823 DCLRE1C-6 + GCGGGCGCCUAGAACCCGAC 20 824 DCLRE1C-7 + GCUUCCCGGAAGUGGCGGCG 20 825 DCLRE1C-8 + GCUUCGAUAGGGAGAACCUG 20 826 DCLRE1C-9 − GGAAGUAGGCGCGGGCCCUC 20 827 DCLRE1C-10 − GGAGACCGGGGGCAAAGUCA 20 828 DCLRE1C-11 − GGAGCAUCCGGUCGGGUUCU 20 829 DCLRE1C-12 + GGCGCGGUCAGGGCUGGCCU 20 830 DCLRE1C-13 + GGCGCUAUGAGUUCUUUCGA 20 831 DCLRE1C-14 + GGCUGCGUUCGGCCGCCCAA 20 832 DCLRE1C-15 − GGGCAAAGUCAAGGAGCAUC 20 833 DCLRE1C-16 + GGGGUCCCGGACUCUGGGAU 20 834 DCLRE1C-17 + GGUCUCCGGACUCCUCUGAU 20 835 DCLRE1C-18 + GGUUUUGGGGUCCCGGACUC 20 836 DCLRE1C-19 + GUCCCGGACUCUGGGAUCGG 20 837 DCLRE1C-20 + GUUUUGGGGUCCCGGACUCU 20 838

Table II.19B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., DCLRE1C gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the DCLRE1C gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the DCLRE1C gene.

TABLE II.19B Exemplary HDR-enhancing gRNAs Targeting a DCLRE1C Gene S. pyogenes A high level of orthogonality 2nd Tier 19B gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID DCLRE1C-21 − AAGCGGUCUAUGGAGAUAGU 20 839 DCLRE1C-22 − ACGCAGCCACGUCCAAUCAG 20 840 DCLRE1C-23 + AGCGCGCGGCUUCCCGGAAG 20 841 DCLRE1C-24 − AUCAGAGGAGUCCGGAGACC 20 842 DCLRE1C-25 − CACGUCCAAUCAGAGGAGUC 20 843 DCLRE1C-26 + CCCAAUGGCGAGGCAGCGCG 20 844 DCLRE1C-27 − CCGCGCGCUGCCUCGCCAUU 20 845 DCLRE1C-28 − CCUGACCGCGCCGCCACUUC 20 846 DCLRE1C-29 − CGCCGCCGAUCCCAGAGUCC 20 847 DCLRE1C-30 + CGGAAGUGGCGGCGCGGUCA 20 848 DCLRE1C-31 + CGGACUCCUCUGAUUGGACG 20 849 DCLRE1C-32 + CGGCGCUAUGAGUUCUUUCG 20 850 DCLRE1C-33 + CGUUCGGCCGCCCAAUGGCG 20 851 DCLRE1C-34 + CUCCAUAGACCGCUUCGAUA 20 852 DCLRE1C-35 − CUCCCUAUCGAAGCGGUCUA 20 853 DCLRE1C-36 − CUGACCGCGCCGCCACUUCC 20 854 DCLRE1C-37 + CUUCGAUAGGGAGAACCUGA 20 855 DCLRE1C-38 − UCAGAGGAGUCCGGAGACCG 20 856 DCLRE1C-39 + UCUCCAUAGACCGCUUCGAU 20 857 DCLRE1C-40 + UGAUUGGACGUGGCUGCGUU 20 858

Table II.20A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., DCLRElC gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the DCLRElC gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the DCLRElC gene.

TABLE II.20A Exemplary HDR-enhancing gRNAs Targeting a DCLRE1C Gene S. aureus A high level of orthogonality, starts with a G, PAM is NNGRRT 1st Tier 20A gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID DCLRE1C-41 − GAACGCAGCCACGUCCAAUCAG 22 859 DCLRE1C-42 − GAACUCAUAGCGCCGCCGAUCC 22 860 DCLRE1C-43 + GCAGCGGGCGCCUAGAACCCGA 22 861 DCLRE1C-44 + GCCUUGGCUUCAGCUGCGGUUU 22 862 DCLRE1C-45 + GCGGUUUUGGGGUCCCGGACUC 22 863 DCLRE1C-46 + GGACUCUGGGAUCGGCGGCGCU 22 864 DCLRE1C-47 − GGCAAAGUCAAGGAGCAUCCGG 22 865

Table II.20B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., DCLRElC gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the DCLRE1C gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the DCLRE1C gene.

TABLE II.20B Exemplary HDR-enhancing gRNAs Targeting a DCLRE1C Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 20B gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID DCLRE1C-48 + AGUUCUUUCGAGGGGCAGAUGG 22 866 DCLRE1C-49 − UCGAAGCGGUCUAUGGAGAUAG 22 867

Table II.21A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., BRCA2 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the BRCA2 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the BRCA2 gene.

TABLE II.21A Exemplary HDR-enhancing gRNAs Targeting a BRCA2 Gene S. pyogenes A high level of orthogonality, and starts with a G 1st Tier 21A gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID BRCA2-1 + GACGGUUGGGAUGCCUGACA 20 868 BRCA2-2 − GAGGCGCAGCAGUGCCACAG 20 869 BRCA2-3 + GCCCACCCAGGCCUGACUUC 20 870 BRCA2-4 + GCCUCGGGUGUCUUUUGCGG 20 871 BRCA2-5 − GCGAAAGGAAAUUCCUUGUC 20 872 BRCA2-6 + GCUGCGCCUCUGCUGCGCCU 20 873 BRCA2-7 − GCUGCGGGUAUUUCUCAGUG 20 874 BRCA2-8 − GUAUUUCUCAGUGUGGCGAA 20 875 BRCA2-9 + GUGUGCUGCGUGUCGCGUCA 20 876

Table II.21B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., BRCA2 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the BRCA2 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the BRCA2 gene.

TABLE II.21B Exemplary HDR-enhancing gRNAs Targeting a BRCA2 Gene S. pyogenes A high level of orthogonality 2nd Tier 21B gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID BRCA2-10 − ACACGCACCACCCGGAAGUC 20 877 BRCA2-11 + ACACUGAGAAAUACCCGCAG 20 878 BRCA2-12 − ACCACCCGGAAGUCAGGCCU 20 879 BRCA2-13 + ACCCAGGCCUGACUUCCGGG 20 880 BRCA2-14 − ACCGCCGCAAAAGACACCCG 20 881 BRCA2-15 + ACGGCGUCACGUGGCCAGCG 20 882 BRCA2-16 + ACGUGGCCAGCGCGGGCUUG 20 883 BRCA2-17 + AUACCCGCAGCGGCCCACCC 20 884 BRCA2-18 − CACCACCCGGAAGUCAGGCC 20 885 BRCA2-19 − CCCGGAAGUCAGGCCUGGGU 20 886 BRCA2-20 + CGCGAGCUUCUGAAACUAGG 20 887 BRCA2-21 + CGGCAGAGGCGGAGCCGCUG 20 888 BRCA2-22 + CGGCGUCACGUGGCCAGCGC 20 889 BRCA2-23 + CGGGUGUCUUUUGCGGCGGU 20 890 BRCA2-24 − CUCGCGCCACAAGCCCGCGC 20 891 BRCA2-25 + CUUCUGAAACUAGGCGGCAG 20 892 BRCA2-26 + UCGGGUGUCUUUUGCGGCGG 20 893 BRCA2-27 + UGCGCCUCGGGUGUCUUUUG 20 894 BRCA2-28 + UGGCGCGAGCUUCUGAAACU 20 895 BRCA2-29 + UGUCGCGUCACGGCGUCACG 20 896

Table II.22A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., BRCA2 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the BRCA2 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the BRCA2 gene.

TABLE II.22A Exemplary HDR-enhancing gRNAs Targeting a BRCA2 Gene S. aureus A high level of orthogonality, starts with a G, PAM is NNGRRT 1st Tier 22A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID BRCA2-30 + GCCUCGGGUGUCUUUUGCGGCG 22 897 BRCA2-31 + GCGGCCCACCCAGGCCUGACUU 22 898

Table II.22B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., BRCA2 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the BRCA2 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the BRCA2 gene.

TABLE II.22B Exemplary HDR-enhancing gRNAs Targeting a BRCA2 Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 22B gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID BRCA2-32 − ACGCACCACCCGGAAGUCAGGC 22 899 BRCA2-33 + ACUGCUGCGCCUCUGCUGCGCC 22 900 BRCA2-34 − AGUCAGGCCUGGGUGGGCCGCU 22 901 BRCA2-35 + CUGACGGUUGGGAUGCCUGACA 22 902

Table II.23A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RAD51 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RAD51 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RAD51 gene.

TABLE II.23A Exemplary HDR-enhancing gRNAs Targeting a RAD51 Gene S. pyogenes A high level of orthogonality, and starts with a G 1st Tier 23A gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID RAD51-1 − GAAGCGCCGCACUCUCCUUA 20 903 RAD51-2 + GAAGGCGGAUCCGGGAGGCG 20 904 RAD51-3 + GAGAAGGCGGAUCCGGGAGG 20 905 RAD51-4 + GCAGGGCGGAAGCGGGGAGA 20 906 RAD51-5 − GCCGCACUCUCCUUAGGGCU 20 907 RAD51-6 + GCGGGAAUUCUGAAAGCCGC 20 908 RAD51-7 + GCUGGGAACUGCAACUCAUC 20 909 RAD51-8 + GCUUCCCGAGGCGUGCAGCU 20 910 RAD51-9 − GGAAGCGCCGCACUCUCCUU 20 911 RAD51-10 + GGAAUUCUGAAAGCCGCUGG 20 912 RAD51-11 + GGAGAGUGCGGCGCUUCCCG 20 913 RAD51-12 + GGCAGUCUGUAAACUCGCGC 20 914 RAD51-13 + GGGAUACGUUACGUCGACGC 20 915 RAD51-14 + GGGCGGAAGCGGGGAGAAGG 20 916 RAD51-15 + GGGCGUGACCCUGGGCGAGA 20 917 RAD51-16 + GGGGAGAAGGCGGAUCCGGG 20 918 RAD51-17 + GGGGAUACGUUACGUCGACG 20 919 RAD51-18 + GUCGACGCGGGCGUGACCCU 20 920 RAD51-19 + GUUAGCGCGCAGGGCGGAAG 20 921

Table II.23B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RAD51 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RAD51 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RAD51 gene.

TABLE II.23B Exemplary HDR-enhancing gRNAs Targeting a RAD51 Gene S. pyogenes A high level of orthogonality 2nd Tier 23B gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID RAD51-20 + AAGCGGGGAGAAGGCGGAUC 20 922 RAD51-21 + AAGCUCUCGAGCUCCCGUCU 20 923 RAD51-22 + AGCUCUCGAGCUCCCGUCUU 20 924 RAD51-23 − AGUUCCCAGCUGCACGCCUC 20 925 RAD51-24 + CCCGUCUUGGGUUAGCGCGC 20 926 RAD51-25 − CCCUGCGCGCUAACCCAAGA 20 927 RAD51-26 − CCGCCAAACCCUCUCGCCCA 20 928 RAD51-27 + CCGUCUUGGGUUAGCGCGCA 20 929 RAD51-28 − CCUGCGCGCUAACCCAAGAC 20 930 RAD51-29 − CGCUGCGCGCGGUCCGCCAG 20 931 RAD51-30 + CGCUGGCGGACCGCGCGCAG 20 932 RAD51-31 + CGGGCGUGACCCUGGGCGAG 20 933 RAD51-32 − CGGUCUCUGGCCGCUGCGCG 20 934 RAD51-33 − CGUAACGUAUCCCCGCCUCC 20 935 RAD51-34 + CGUCGACGCGGGCGUGACCC 20 936 RAD51-35 + UAGCGCGCAGGGCGGAAGCG 20 937 RAD51-36 + UCAUCUGGGUUGUGCGCAGA 20 938 RAD51-37 + UCUUGGGUUAGCGCGCAGGG 20 939 RAD51-38 + UGGGUUGUGCGCAGAAGGCU 20 940 RAD51-39 + UUAGCGCGCAGGGCGGAAGC 20 941

Table II.24A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RAD51 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RAD51 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RAD51 gene.

TABLE II.24A Exemplary HDR-enhancing gRNAs Targeting a RAD51 Gene S. aureus A high level of orthogonality, starts with a G, PAM is NNGRRT 1st Tier 24A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID RAD51-40 − GAGCUCGAGAGCUUGAUCCUGC 22 942 RAD51-41 + GAGGGCAGUCUGUAAACUCGCG 22 943 RAD51-42 + GCAGCUGGGAACUGCAACUCAU 22 944 RAD51-43 + GCAGGGCGGAAGCGGGGAGAAG 22 945 RAD51-44 + GGAGAAGGCGGAUCCGGGAGGC 22 946

Table II.24B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RAD51 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RAD51 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RAD51 gene.

TABLE II.24B Exemplary HDR-enhancing gRNAs Targeting a RAD51 Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 24B gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID RAD51-45 + ACCCUGGGCGAGAGGGUUUGGC 22 947 RAD51-46 + ACGCGGGCGUGACCCUGGGCGA 22 948 RAD51-47 + AUCAAGCUCUCGAGCUCCCGUC 22 949 RAD51-48 − AUUCCCGCCAAACCCUCUCGCC 22 950 RAD51-49 − CAGCCUUCUGCGCACAACCCAG 22 951 RAD51-50 − CGACGUAACGUAUCCCCGCCUC 22 952 RAD51-51 − CGCGCGGUCCGCCAGCGGCUUU 22 953 RAD51-52 + CGGCCAGAGACCGAGCCCUAAG 22 954

Table II.25A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., XRCC1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the XRCC1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the XRCC1 gene.

TABLE II.25A Exemplary HDR-enhancing gRNAs Targeting a XRCC1 Gene S. pyogenes A high level of orthogonality, and starts with a G 1st Tier 25A gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID XRCC1-1 − GAAGGAUGAGGUAGAGUAUG 20 955 XRCC1-2 − GACAUGGGGUGAGAGGGCGG 20 956 XRCC1-3 + GACGCCGGCGCCGGCGCGCC 20 957 XRCC1-4 − GACGUCCGAACCCUGCUUUC 20 958 XRCC1-5 − GAGGUAGAGUAUGGGGUCCG 20 959 XRCC1-6 − GAGUAUGGGGUCCGAGGGGC 20 960 XRCC1-7 + GCGCUCUUCCCGCUCUGGAG 20 961 XRCC1-8 + GCGGGGUUGUGUGUGGCGGA 20 962 XRCC1-9 + GGAGGAAACGCUCGUUGCUA 20 963 XRCC1-10 + GGCUAGAGCGGGGUUGUGUG 20 964 XRCC1-11 + GGCUCCCAGAAAGCAGGGUU 20 965 XRCC1-12 − GGCUCGGGCCUUUCAAACCC 20 966 XRCC1-13 + GGCUUGCGCAGUGUCGACGC 20 967 XRCC1-14 − GGGCGGGGUGCGCCCUGCGC 20 968 XRCC1-15 + GGGUUGUGUGUGGCGGAGGG 20 969 XRCC1-16 − GGUAGAGUAUGGGGUCCGAG 20 970 XRCC1-17 − GGUCCGAGGGGCAGGGAGAG 20 971 XRCC1-18 − GUCCGAGGGGCAGGGAGAGU 20 972 XRCC1-19 − GUGCGCAAGCGCGCGAGGCU 20 973 XRCC1-20 − GUGGGCUUCGCCUGGCCAGA 20 974

Table II.25B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., XRCC1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the XRCC1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the XRCC1 gene.

TABLE II.25B Exemplary HDR-enhancing gRNAs Targeting a XRCC1 Gene S. pyogenes A high level of orthogonality 2nd Tier 25B gRNA Target Site Seq Name  DNA Strand Targeting Domain Length ID XRCC1-21 + AAGCAGGGUUCGGACGUCAU 20 975 XRCC1-22 − ACAUGGCGGAGGCGGAUCUC 20 976 XRCC1-23 + ACGCAGCGCUCUUCCCGCUC 20 977 XRCC1-24 − ACGUCCGAACCCUGCUUUCU 20 978 XRCC1-25 + ACUCCAUCGUGCAAUGAGAA 20 979 XRCC1-26 + AGCAGGGUUCGGACGUCAUU 20 980 XRCC1-27 + AGGGUUCGGACGUCAUUGGG 20 981 XRCC1-28 − CAGUCGCGCCUCUCCAGAGC 20 982 XRCC1-29 + CCGCUCUGGAGAGGCGCGAC 20 983 XRCC1-30 + CCGGCGCGCCGGGGUUUGAA 20 984 XRCC1-31 − CCUUUCAAACCCCGGCGCGC 20 985 XRCC1-32 − CGACCUCCGGGAUUGGUGUC 20 986 XRCC1-33 + CGCUCUGGAGAGGCGCGACU 20 987 XRCC1-34 − CUCCGGCAUGUCAACGUCGU 20 988 XRCC1-35 − UCAACGUCGUGGGCUUCGCC 20 989 XRCC1-36 + UCGGACGUCAUUGGGAGGCG 20 990 XRCC1-37 − UCUCCGGCAUGUCAACGUCG 20 991 XRCC1-38 − UGCGCAAGCGCGCGAGGCUC 20 992 XRCC1-39 + UGCGCACUUUAGCCAGCGCA 20 993 XRCC1-40 + UUGCGCACUUUAGCCAGCGC 20 994

Table II.26A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., XRCC1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the XRCC1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the XRCC1 gene.

TABLE II.26A Exemplary HDR-enhancing gRNAs Targeting a XRCC1 Gene S. aureus A high level of orthogonality, starts with a G, PAM is NNGRRT 1st Tier 26A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID XRCC1-41 − GACAGGGUCUUGCUCUCUCACC 22  995 XRCC1-42 + GAUCGUGCCACUGCACUCCAUC 22  996 XRCC1-43 − GCCAGAAGGAUGAGGUAGAGUA 22  997 XRCC1-44 − GCCUAGCAACAGAAGCGACCUC 22  998 XRCC1-45 + GCUACUUAGGAGGCUGAAGUGG 22  999 XRCC1-46 + GGAUCCCUUGGCCCCAGGAGAC 22 1000 XRCC1-47 − GGGCAGGGAGAGUGGGAGGGGG 22 1001 XRCC1-48 − GGUCUUGCUCUCUCACCCAGGA 22 1002 XRCC1-49 + GUCGACGCCGGCGCCGGCGCGC 22 1003 XRCC1-50 − GUCGUGGGCUUCGCCUGGCCAG 22 1004

Table II.26B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., XRCC1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the XRCC1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the XRCC1 gene.

TABLE II.26B Exemplary HDR-enhancing gRNAs Targeting a XRCC1 Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 26B gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID XRCC1-51 + ACUCCAUCGUGCAAUGAGAAAG 22 1005 XRCC1-52 − AGUAUGGGGUCCGAGGGGCAGG 22 1006 XRCC1-53 − AUUCGCCUUUCUCAUUGCACGA 22 1007 XRCC1-54 − CAACCCCUGUCUCCUGGGGCCA 22 1008 XRCC1-55 + CCACAAAAAAUACAAAAAUUAG 22 1009 XRCC1-56 − CCACUUCAGCCUCCUAAGUAGC 22 1010 XRCC1-57 + CUGUUGCUAGGCUCCCAGAAAG 22 1011 XRCC1-58 + UCAUUGGGAGGCGAGGCUAGAG 22 1012 XRCC1-59 − UCGCCUGGCCAGAAGGAUGAGG 22 1013 XRCC1-60 − UUUUAAAAAUUUUUUGUUGAGA 22 1014 XRCC1-61 − UUUUGUAUUUUUUGUGGAGACA 22 1015

Table II.27A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., LIG1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the LIG1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the LIG1 gene.

TABLE II.27A Exemplary HDR-enhancing gRNAs Targeting a LIG1 Gene S. pyogenes A high level of orthogonality, and starts with a G 1st Tier 27A gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID LIG1-1 − GACGUCUGCGGGCGGGGGCG 20 1016 LIG1-2 + GACUGCAGAGGCGCGCCUGG 20 1017 LIG1-3 − GCAACACACUCAGAUCCGCC 20 1018 LIG1-4 − GCAGUCCCAAGUUCGCGCCA 20 1019 LIG1-5 − GCCCGCGCUUUCCCUCGCCC 20 1020 LIG1-6 − GCCGUCGCGCGGAGGACACU 20 1021 LIG1-7 + GCCUAUGCUUCGCCAUGUCG 20 1022 LIG1-8 + GCGCGAACUUGGGACUGCAG 20 1023 LIG1-9 − GCGCGCAGACGUCUGCGGGC 20 1024 LIG1-10 − GCGGGGCAUCCCGGGAGCAA 20 1025 LIG1-11 − GGAGACCGCGCGGGGCAUCC 20 1026 LIG1-12 − GGAGUCGUAGUCUCCCGAAU 20 1027 LIG1-13 + GGCCUAUGCUUCGCCAUGUC 20 1028 LIG1-14 + GGCGGGUGCGCCGAAUGCUU 20 1029 LIG1-15 + GGGACCAACGCAAGGCAAGU 20 1030 LIG1-16 − GGGAGUCGUAGUCUCCCGAA 20 1031 LIG1-17 + GGGCCUAUGCUUCGCCAUGU 20 1032 LIG1-18 + GGGGCCGUCCGCAAGCAGAU 20 1033 LIG1-19 − GGUCUGAGGAGUGACUGGCA 20 1034 LIG1-20 − GUCGUAGUCUCCCGAAUGGG 20 1035

Table II.27B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., LIG1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the LIG1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the LIG1 gene.

TABLE II.27B Exemplary HDR-enhancing gRNAs Targeting a LIG1 Gene S. pyogenes A high level of orthogonality 2nd Tier 27B gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID LIG1-21 + ACACCCGCUCAUCCAGGGCG 20 1036 LIG1-22 + ACGUCUGCGCGCGAAUGCCG 20 1037 LIG1-23 − AUUCGCGCGCAGACGUCUGC 20 1038 LIG1-24 + CACCCGCUCAUCCAGGGCGA 20 1039 LIG1-25 + CAGUGUCCUCCGCGCGACGG 20 1040 LIG1-26 − CAUUCGCGCGCAGACGUCUG 20 1041 LIG1-27 − CGCCGUCGCGCGGAGGACAC 20 1042 LIG1-28 − CGCGCGCAGACGUCUGCGGG 20 1043 LIG1-29 + CGGCGCGCGGGACCAACGCA 20 1044 LIG1-30 + CGGCGGGUGCGCCGAAUGCU 20 1045 LIG1-31 + CGGCGGUGCGGACGGUGCCC 20 1046 LIG1-32 + CGGGACCAACGCAAGGCAAG 20 1047 LIG1-33 + UCCCAGUGUCCUCCGCGCGA 20 1048 LIG1-34 + UCCGCGCGACGGCGGCGGUG 20 1049 LIG1-35 − UCGCCCUGGAUGAGCGGGUG 20 1050 LIG1-36 − UCGGUGGAAGCGCCCCCGCG 20 1051 LIG1-37 + UCUCUUCCCGCCGUGCCUCG 20 1052 LIG1-38 + UCUUCCCGCCGUGCCUCGCG 20 1053 LIG1-39 + UGUCCUCCGCGCGACGGCGG 20 1054 LIG1-40 − UUCCCUCGCCCUGGAUGAGC 20 1055

Table II.28A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., LIG1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the LIG1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the LIG1 gene.

TABLE II.28A Exemplary HDR-enhancing gRNAs Targeting a LIG1 Gene S. aureus A high level of orthogonality, starts with a G, PAM is NNGRRT 1st Tier 28A gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID LIG1-41 + GAGGCGGAGGGCGGCGGGUGCG 22 1056 LIG1-42 − GAGUGACUGGCAGGGAAAGAGG 22 1057 LIG1-43 − GGACACUGGGAGUCGUAGUCUC 22 1058 LIG1-44 + GGCCUGGCCCGGCCCUUGCUCC 22 1059

Table II.28B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., LIG1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the LIG1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the LIG1 gene.

TABLE II.28B Exemplary HDR-enhancing gRNAs Targeting a LIG1 Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 28B gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID LIG1-45 − AAUGCCCGCGCUUUCCCUCGCC 22 1060 LIG1-46 + AGCAGAUGGGAGGCGGAGGGCG 22 1061 LIG1-47 − AGCGGGUGUGGCUGAGGGUCUG 22 1062 LIG1-48 + CCCCGCCCGCAGACGUCUGCGC 22 1063 LIG1-49 − CCCUGGAUGAGCGGGUGUGGCU 22 1064 LIG1-50 − CCGCCGUCGCGCGGAGGACACU 22 1065 LIG1-51 − CGAAUGGGAGGAGGGCGGGAAA 22 1066 LIG1-52 + CGCCAUGUCGGGGUGUCUGCAG 22 1067 LIG1-53 − CGCUUUCCCUCGCCCUGGAUGA 22 1068 LIG1-54 + UGCAGAGGCGCGCCUGGCGGAU 22 1069 LIG1-55 + UGGGACUGCAGAGGCGCGCCUG 22 1070 LIG1-56 + UGGGGCCUAUGCUUCGCCAUGU 22 1071

Table II.29A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., LIG3 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the LIG3 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the LIG3 gene.

TABLE II.29A Exemplary HDR-enhancing gRNAs Targeting a LIG3 Gene S. pyogenes A high level of orthogonality, and starts with a G 1st Tier  29A gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID LIG3-1 + GAAUGCAACUACGACCCACG 20 1072 LIG3-2 + GACAGGCGCUCCAACCGUCG 20 1073 LIG3-3 + GAGCCGGAGAGGCAGGUGAG 20 1074 LIG3-4 + GAGGCAGGUGAGGGGCUACG 20 1075 LIG3-5 − GCGCCUGUCUCUUUAAAUCC 20 1076 LIG3-6 − GCGCGCAGGCGCAAGAGCCA 20 1077 LIG3-7 + GGACCCGGAUUUAAAGAGAC 20 1078 LIG3-8 + GGAGCCGGAGAGGCAGGUGA 20 1079 LIG3-9 + GGGGACCGGUCGCGUGGCCG 20 1080 LIG3-10 + GGGGGCGGGGACCGGUCGCG 20 1081 LIG3-11 + GGUGAGCGCCGGAGCCGGAG 20 1082

Table II.29B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., LIG3 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the LIG3 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the LIG3 gene.

TABLE II.29B Exemplary HDR-enhancing gRNAs Targeting a LIG3 Gene S. pyogenes  A high level of orthogonality 2nd Tier 29B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID LIG3-12 − AACUUGCUCAUUACAGGCCG 20 1083 LIG3-13 − AAUCCGGGUCCUAGAGCGGG 20 1084 LIG3-14 + ACAGGCGCUCCAACCGUCGU 20 1085 LIG3-15 + ACUACGACCCACGUGGCAGA 20 1086 LIG3-16 − ACUUGCUCAUUACAGGCCGC 20 1087 LIG3-17 + CAACCGUCGUGGGCUGCCCG 20 1088 LIG3-18 − CAAGGCCGCGGCCACGCGAC 20 1089 LIG3-19 − CCGGCGCUCACCGUAGGCCU 20 1090 LIG3-20 − CCGGCUCCGGCGCUCACCGU 20 1091 LIG3-21 + CCUACGGUGAGCGCCGGAGC 20 1092 LIG3-22 − CCUCGGAACUUGCUCAUUAC 20 1093 LIG3-23 + CCUGUAAUGAGCAAGUUCCG 20 1094 LIG3-24 − CGCGGGCAGCCCACGACGGU 20 1095 LIG3-25 + CGGUCGCGUGGCCGCGGCCU 20 1096 LIG3-26 + CUACGACCCACGUGGCAGAC 20 1097 LIG3-27 + UACGACCCACGUGGCAGACG 20 1098 LIG3-28 − UAGAGCGGGAGGCAGCGCGC 20 1099 LIG3-29 + UGAGCAAGUUCCGAGGCCUA 20 1100 LIG3-30 − UUAAAUCCGGGUCCUAGAGC 20 1101 LIG3-31 − UUUAAAUCCGGGUCCUAGAG 20 1102

Table II.30A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., LIG3 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the LIG3 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the LIG3 gene.

TABLE II.30A Exemplary HDR-enhancing gRNAs Targeting a LIG3 Gene S. aureus  A high level of orthogonality, starts with a G, PAM is NNGRRT 1st Tier 30A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID LIG3-32 + GCGCUGCCUCCCGCUCUAGGAC 22 1103 LIG3-33 − GGUCCCCGCCCCCGUCUGCCAC 22 1104

Table II.30B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., LIG3 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the LIG3 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the LIG3 gene.

TABLE II.30B Exemplary HDR-enhancing gRNAs Targeting a LIG3 Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 30B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID LIG3-34 + CUCCCAAACAUCACAGGGCAGG 22 1105 LIG3-35 − UGGAGCGCCUGUCUCUUUAAAU 22 1106 LIG3-36 − UUCUGCCUGCCCUGUGAUGUUU 22 1107

Table II.31A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., POLQ gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the POLQ gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the POLQ gene.

TABLE II.31A Exemplary HDR-enhancing gRNAs Targeting a POLQ Gene S. pyogenes A high level of orthogonality, and starts with a G 1st Tier 31A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID POLQ-1 + GAACUCUAUGGUUCCGGGGC 20 1108 POLQ-2 + GAGGGAGGACGCUGGGACUG 20 1109 POLQ-3 + GCUCCUUCCCCACGAGUCUA 20 1110 POLQ-4 + GGACUGUGGCUUGCCCUGAU 20 1111 POLQ-5 + GGAGGUUUGAGUUUGAAGAC 20 1112 POLQ-6 − GGGGAAGGAGCGGCUCUCGC 20 1113 POLQ-7 + GGUUUGAGUUUGAAGACUGG 20 1114 POLQ-8 − GUCCCAGCGUCCUCCCUCUC 20 1115 POLQ-9 − GUCUUCAAACUCAAACCUCC 20 1116 POLQ-10 + GUUUGAGUUUGAAGACUGGC 20 1117

Table II.31B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., POLQ gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the POLQ gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the POLQ gene.

TABLE II.31B Exemplary HDR-enhancing gRNAs Targeting a POLQ Gene S. pyogenes A high level of orthogonality 2nd Tier 31B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID POLQ-11 − AAGCCAUAGACUCGUGGGGA 20 1118 POLQ-12 − ACCCGAAAGCCAUAGACUCG 20 1119 POLQ-13 + AGAACUCUAUGGUUCCGGGG 20 1120 POLQ-14 + AGGCCAGGGUUCUCCCGAGA 20 1121 POLQ-15 + CACGGAGAACUCUAUGGUUC 20 1122 POLQ-16 + CAGGCCAGGGUUCUCCCGAG 20 1123 POLQ-17 + CCAGGGUUCUCCCGAGAGGG 20 1124 POLQ-18 + CCCACGAGUCUAUGGCUUUC 20 1125 POLQ-19 + CCCCACGAGUCUAUGGCUUU 20 1126 POLQ-20 − CCCGAAAGCCAUAGACUCGU 20 1127 POLQ-21 − CCGAAAGCCAUAGACUCGUG 20 1128 POLQ-22 − CGGAACCAUAGAGUUCUCCG 20 1129 POLQ-23 + CGGAGAACUCUAUGGUUCCG 20 1130 POLQ-24 + CUAUGGUUCCGGGGCGGGCC 20 1131 POLQ-25 + CUCCCGAGAGGGAGGACGCU 20 1132 POLQ-26 − UCAAACCUCCCGGCCCGCCC 20 1133 POLQ-27 − UCGCUGGCGUCUAAGACUUC 20 1134 POLQ-28 + UCUAUGGUUCCGGGGCGGGC 20 1135 POLQ-29 + UCUCCCGAGAGGGAGGACGC 20 1136 POLQ-30 + UUAAGCCACGGAGAACUCUA 20 1137

Table II.32A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., POLQ gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the POLQ gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the POLQ gene.

TABLE II.32A Exemplary HDR-enhancing gRNA Targeting a POLQ Gene S. aureus A high level of orthogonality, starts with a G, PAM is NNGRRT 1st Tier 32A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID POLQ-31 + GUUCCGGGGCGGGCCGGGAGGU 22 1138

Table II.32B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., POLQ gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the POLQ gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the POLQ gene.

TABLE II.32B Exemplary HDR-enhancing gRNAs Targeting a POLQ Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 32B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID POLQ-32 + AUGUCCGCAGCUGUUGCCAGGC 22 1139 POLQ-33 + CAGCGAGAGCCGCUCCUUCCCC 22 1140 POLQ-34 − CUCCCGGCCCGCCCCGGAACCA 22 1141 POLQ-35 + CUUCCCCACGAGUCUAUGGCUU 22 1142 POLQ-36 + UGGCUUGCCCUGAUCGGCCGAG 22 1143

Table II.33A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., FBXO18 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the FBX18 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the FBXO18 gene.

TABLE II.33A Exemplary HDR-enhancing gRNAs Targeting a FBXO18 Gene S. pyogenes A high level of orthogonality, and starts with a G 1st Tier 33A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID FBXO18-1 − GAAGCGCCCGCCGCCGGAGC 20 1144 FBXO18-2 − GACCAAUCGGGACGCGAGAC 20 1145 FBXO18-3 − GACCGGAGGGGGCGUGCUGA 20 1146 FBXO18-4 − GACGGCCCCCGCGACCAAUC 20 1147 FBXO18-5 + GAGCUCGCGGAGGAAGUCGG 20 1148 FBXO18-6 + GAGGAAGUCGGCGGGCGUCU 20 1149 FBXO18-7 − GCACUGUGGCGCUCCGGACC 20 1150 FBXO18-8 + GCGGAGCUCGCGGAGGAAGU 20 1151 FBXO18-9 − GGACCCCCCGCGCAGGACCC 20 1152 FBXO18-10 − GGAGGGGGCGUGCUGACGGA 20 1153 FBXO18-11 + GGCCGUCAGUCCGGCUCCGG 20 1154 FBXO18-12 + GGGACGCUGGGCUGAGCGGC 20 1155 FBXO18-13 + GGGGGCCGUCAGUCCGGCUC 20 1156 FBXO18-14 + GGGUCCUGCGCGGGGGGUCC 20 1157 FBXO18-15 + GGUCGCGGGGGCCGUCAGUC 20 1158 FBXO18-16 + GUCAGUCCGGCUCCGGCGGC 20 1159 FBXO18-17 + GUCCGUCAGCACGCCCCCUC 20 1160 FBXO18-18 − GUCUGCGGCCUCACGCACUG 20 1161 FBXO18-19 + GUGAGGCCGCAGACGUGGCA 20 1162 FBXO18-20 + GUGGGAGGGGCUCCGCCGUG 20 1163

Table II.33B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., FBXO18 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the FBXO18 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the FBXO18 gene.

TABLE II.33B Exemplary HDR-enhancing gRNAs Targeting a FBXO18 Gene S. pyogenes A high level of orthogonality 2nd Tier 33B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID FBXO18-21 + AACCUCCGGGGUCCUGCGCG 20 1164 FBXO18-22 − AAUCGGGACGCGAGACCGGA 20 1165 FBXO18-23 + ACCUCCGGGGUCCUGCGCGG 20 1166 FBXO18-24 + AGAGGAGGAGCUCGCUGCCG 20 1167 FBXO18-25 + AGCUCGCGGAGGAAGUCGGC 20 1168 FBXO18-26 + AGGAAGUCGGCGGGCGUCUC 20 1169 FBXO18-27 + AGUGCGUGAGGCCGCAGACG 20 1170 FBXO18-28 − AUCGGGACGCGAGACCGGAG 20 1171 FBXO18-29 − CAAUCGGGACGCGAGACCGG 20 1172 FBXO18-30 − CGCCGCCGGAGCCGGACUGA 20 1173 FBXO18-31 + CGCGUCCCGAUUGGUCGCGG 20 1174 FBXO18-32 + CGGCGGGCGUCUCGGGCUCC 20 1175 FBXO18-33 + CGUCAGUCCGGCUCCGGCGG 20 1176 FBXO18-34 + CUCCGGUCUCGCGUCCCGAU 20 1177 FBXO18-35 + CUCGCGUCCCGAUUGGUCGC 20 1178 FBXO18-36 + UAACCUCCGGGGUCCUGCGC 20 1179 FBXO18-37 + UCGCGUCCCGAUUGGUCGCG 20 1180 FBXO18-38 + UCUCGCGUCCCGAUUGGUCG 20 1181 FBXO18-39 − UGACGGCCCCCGCGACCAAU 20 1182 FBXO18-40 + UUAACCUCCGGGGUCCUGCG 20 1183

Table II.34A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., FBXO18 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the FBXO18 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the FBXO18 gene.

TABLE II.34A Exemplary HDR-enhancing gRNAs Targeting a FBXO18 Gene S. aureus A high level of orthogonality, starts with a G, PAM is NNGRRT 1st Tier 34A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID FBXO18-41 + GCCGUGUGGAAAACUUAACCUC 22 1184 FBXO18-42 + GCGGGCCCGGCGGCGGCGGCAG 22 1185 FBXO18-43 + GCGUGAGGCCGCAGACGUGGCA 22 1186

Table II.34B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., FBXO18 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the FBXO18 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the FBXO18 gene.

TABLE II.34B Exemplary HDR-enhancing gRNAs Targeting a FBXO18 Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 34B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID FBXO18-44 + CCGGCGGCGGCGGCAGCGGGGU 22 1187 FBXO18-45 + UCCUGCGCGGGGGGUCCGGGCC 22 1188 FBXO18-46 + UUAACCUCCGGGGUCCUGCGCG 22 1189

Table II.35A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RTEL1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RTEL1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RTEL1 gene.

TABLE II.35A Exemplary HDR-enhancing gRNAs Targeting a RTEL1 Gene S. pyogenes A high level of orthogonality, and starts with a G 1st Tier 35A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID RTEL1-1 − GAAACUGUUCCCCCGCGGAG 20 1190 RTEL1-2 + GAACGCGCAAAACGCCGUGU 20 1191 RTEL1-3 + GACGGGUGGCGGCCCUCGAC 20 1192 RTEL1-4 + GAGCAGGCGGACCCCCUCCG 20 1193 RTEL1-5 − GAGGGGGUCCGCCUGCUCUU 20 1194 RTEL1-6 − GCACUUCCGCCCCCCACUUC 20 1195 RTEL1-7 + GCAGGCGGACCCCCUCCGCG 20 1196 RTEL1-8 − GCCUGCUCUUCGGCUCCUCC 20 1197 RTEL1-9 + GCCUGGAGGAGCCGAAGAGC 20 1198 RTEL1-10 − GCGGCGAACCUUCCAGAACC 20 1199 RTEL1-11 + GCGGGGGAACAGUUUCCGCC 20 1200 RTEL1-12 + GCUGGCUGACAGCUGGGGAC 20 1201 RTEL1-13 − GGAAACUGUUCCCCCGCGGA 20 1202 RTEL1-14 + GGAGUCGGUUGAGUUCCUGA 20 1203 RTEL1-15 + GGCUGACAGCUGGGGACGGG 20 1204 RTEL1-16 + GGGAGCACAAAGCAACGGAC 20 1205 RTEL1-17 − GGUCCGUUGCUUUGUGCUCC 20 1206 RTEL1-18 + GUGGGGGGCGGAAGUGCAGU 20 1207 RTEL1-19 + GUUGAGUUCCUGAGGGACCC 20 1208

Table II.35B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RTEL1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RTEL1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RTEL1 gene.

TABLE II.35B Exemplary HDR-enhancing gRNAs Targeting a RTEL1 Gene S. pyogenes A high level of orthogonality 2nd Tier 35B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID RTEL1-20 + AAAACGCCGUGUAGGCCUGG 20 1209 RTEL1-21 − AAACUGUUCCCCCGCGGAGG 20 1210 RTEL1-22 + AAAGCAACGGACCGGAAGUG 20 1211 RTEL1-23 + AACGGACCGGAAGUGGGGGG 20 1212 RTEL1-24 − AACUCAACCGACUCCAGUCG 20 1213 RTEL1-25 + ACAAAGCAACGGACCGGAAG 20 1214 RTEL1-26 − ACUCAACCGACUCCAGUCGA 20 1215 RTEL1-27 + ACUCUGAGCUGGCUGACAGC 20 1216 RTEL1-28 + AGCAACGGACCGGAAGUGGG 20 1217 RTEL1-29 − AGCCAGCUCAGAGUUUUCGC 20 1218 RTEL1-30 + CAAAGCAACGGACCGGAAGU 20 1219 RTEL1-31 + CGCAAAACGCCGUGUAGGCC 20 1220 RTEL1-32 − CGCGGCGAACCUUCCAGAAC 20 1221 RTEL1-33 + CGCGGGGGAACAGUUUCCGC 20 1222 RTEL1-34 − CGGAAACUGUUCCCCCGCGG 20 1223 RTEL1-35 − CGGCGGAAACUGUUCCCCCG 20 1224 RTEL1-36 + CGGUUCUGGAAGGUUCGCCG 20 1225 RTEL1-37 − CGUUUUGCGCGUUCUGUGUC 20 1226 RTEL1-38 + UGCCCGCGAAAACUCUGAGC 20 1227 RTEL1-39 + UUCCUGAGGGACCCCGGUUC 20 1228

Table II.36A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RTEL1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RTEL1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RTEL1 gene.

TABLE II.36A Exemplary HDR-enhancing gRNAs Targeting a RTEL1 Gene S. aureus A high level of orthogonality, starts with a G, PAM is NNGRRT 1st Tier 36A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID RTEL1-40 − GCGGAAACUGUUCCCCCGCGGA 22 1229 RTEL1-41 + GGCGGCCCUCGACUGGAGUCGG 22 1230 RTEL1-42 + GGGACGGGUGGCGGCCCUCGAC 22 1231

Table II.36B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RTEL1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RTEL1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RTEL1 gene.

TABLE II.36B Exemplary HDR-enhancing gRNAs Targeting a RTEL1 Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 36B Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID RTEL1-43 − CGUCCCCAGCUGUCAGCCAGCU 22 1232 RTEL1-44 + CUGAGCUGGCUGACAGCUGGGG 22 1233 RTEL1-45 − UCCGCGGCGAACCUUCCAGAAC 22 1234

Table II.37A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., PARPBP gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the PARPBP gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the PARPBP gene.

TABLE II.37A Exemplary HDR-enhancing gRNAs Targeting a PARPBP Gene A high level of orthogonality, and starts with a G S. pyogenes 37A 1st Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID PARPBP-1 − GAAUACAGUUCAAACCUCGC 20 1235 PARPBP-2 − GACGCGAGACUUACGUGAUU 20 1236 PARPBP-3 + GAGCGCAGCGAUUGGCUCCC 20 1237 PARPBP-4 + GAGGCAGGCUGGUCUUCCUU 20 1238 PARPBP-5 + GAGGUUUGAACUGUAUUCAG 20 1239 PARPBP-6 + GCAUUUUUAAGUGGUGAUUC 20 1240 PARPBP-7 + GCCGCGGGAGGGCAUCCCGU 20 1241 PARPBP-8 + GCGACUGCGGCGGCCGCGGG 20 1242 PARPBP-9 − GCGCGUCGCGGCAGCCCCCA 20 1243 PARPBP-10 + GCGGCGACUGCGGCGGCCGC 20 1244 PARPBP-11 − GCUGCGCUCGCCCUCCGACC 20 1245 PARPBP-12 + GGCAGGCUGGUCUUCCUUGG 20 1246 PARPBP-13 + GGCGACAGCGGCGACUGCGG 20 1247 PARPBP-14 + GGCUCCCGGGGCCUCCCGCG 20 1248 PARPBP-15 + GGCUCUGCUUCCGGGUCGGA 20 1249 PARPBP-16 + GGGGCUGCCGCGACGCGCUG 20 1250 PARPBP-17 − GUGUGCGGAAGGAUCCCCAA 20 1251

Table II.37B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., PARPBP gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the PARPBP gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the PARPBP gene.

TABLE II.37B Exemplary HDR-enhancing gRNAs Targeting a PARPBP Gene A high level of orthogonality S. pyogenes 37B 2nd Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID PARPBP-18 − AAGACGUACUCUUCAGUGUG 20 1252 PARPBP-19 − ACGCGAGACUUACGUGAUUA 20 1253 PARPBP-20 + ACGCGCUGUGGCUCUGCUUC 20 1254 PARPBP-21 + ACUGUAUUCAGCGGCGACAG 20 1255 PARPBP-22 − AGCAGAGCCACAGCGCGUCG 20 1256 PARPBP-23 + AGCGCAGCGAUUGGCUCCCG 20 1257 PARPBP-24 + CACACUGAAGAGUACGUCUU 20 1258 PARPBP-25 − CCCAACGGGAUGCCCUCCCG 20 1259 PARPBP-26 + CCGCGGGAGGGCAUCCCGUU 20 1260 PARPBP-27 + CGAGCGCAGCGAUUGGCUCC 20 1261 PARPBP-28 − CGCGAGACUUACGUGAUUAG 20 1262 PARPBP-29 + CGCGGGAGGGCAUCCCGUUG 20 1263 PARPBP-30 + CGGAGGGCGAGCGCAGCGAU 20 1264 PARPBP-31 − CGUACUCUUCAGUGUGCGGA 20 1265 PARPBP-32 − UACAGUUCAAACCUCGCGGG 20 1266 PARPBP-33 − UCAAACCUCGCGGGAGGCCC 20 1267 PARPBP-34 − UCACCACUUAAAAAUGCGAC 20 1268 PARPBP-35 − UGAAUACAGUUCAAACCUCG 20 1269 PARPBP-36 + UGCCCUGUCGCAUUUUUAAG 20 1270 PARPBP-37 − UGUGCGGAAGGAUCCCCAAC 20 1271

Table II.38A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., PARPBP gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the PARPBP gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the PARPBP gene.

TABLE II.38A Exemplary HDR-enhancing gRNAs Targeting a PARPBP Gene A high level of orthogonality, starts with a G, PAM is NNGRRT S. aureus 38A 1st Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID PARPBP-38 − GCCGCAGUCGCCGCUGUCGCCG 22 1272 PARPBP-39 + GCGACGCGCUGUGGCUCUGCUU 22 1273 PARPBP-40 + GGCCGCGGGAGGGCAUCCCGUU 22 1274

Table II.38B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., PARPBP gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the PARPBP gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the PARPBP gene.

TABLE II.38B Exemplary HDR-enhancing gRNAs Targeting a PARPBP Gene A high level of orthogonality, and PAM is NNGRRT S. aureus 38B 2nd Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID PARPBP-41 − AGACGUACUCUUCAGUGUGCGG 22 1275 PARPBP-42 − CAGACGCGAGACUUACGUGAUU 22 1276 PARPBP-43 − CAGUGUGCGGAAGGAUCCCCAA 22 1277 PARPBP-44 + CCGCACACUGAAGAGUACGUCU 22 1278 PARPBP-45 + UUGGGGAUCCUUCCGCACACUG 22 1279

Table II.39A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., UIMC1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the UIMC1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the UIMC1 gene.

TABLE II.39A Exemplary HDR-enhancing gRNAs Targeting a UIMC1 Gene A high level of orthogonality, and starts with a G S. pyogenes 39A 1st Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID UIMC1-1 − GAAUCCGCCCCGGAAUCGGG 20 1280 UIMC1-2 + GACCGGCCAUUACUGGUGCC 20 1281 UIMC1-3 − GACUUAACCAACCCCCGCCG 20 1282 UIMC1-4 − GCACCAGUAAUGGCCGGUCC 20 1283 UIMC1-5 − GCCACACGUUGGGAGCGCGG 20 1284 UIMC1-6 − GCCGCCACACGUUGGGAGCG 20 1285 UIMC1-7 − GCGUCGCGAGAGACACACCC 20 1286 UIMC1-8 − GGACGUACCAACUGCGCGGG 20 1287 UIMC1-9 − GGCGGCGGGUACUCACUCGC 20 1288 UIMC1-10 + GGGGUGUGUCUCUCGCGACG 20 1289 UIMC1-11 + GGGUGUGUCUCUCGCGACGC 20 1290 UIMC1-12 − GGUCGCGAGCCGCCACACGU 20 1291 UIMC1-13 + GGUGUGUCUCUCGCGACGCG 20 1292 UIMC1-14 − GUAGACCUUCUCCGGGUUGC 20 1293 UIMC1-15 + GUCCCUCCGGACGCCGAAGU 20 1294 UIMC1-16 + GUCCGCGGCCCGCUACUCUC 20 1295 UIMC1-17 − GUCGCGAGCCGCCACACGUU 20 1296 UIMC1-18 + GUCUCUCGCGACGCGGGGGU 20 1297 UIMC1-19 + GUGUGGCGGCUCGCGACCCC 20 1298 UIMC1-20 + GUGUGUCUCUCGCGACGCGG 20 1299

Table II.39B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., UIMC1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the UIMC1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site TS S) of the UIMC1 gene.

TABLE II.39B Exemplary HDR-enhancing gRNAs Targeting a UIMC1 Gene A high level of orthogonality S. pyogenes 398 2nd Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID UIMC1-21 − AACCCGGCACCAGUAAUGGC 20 1300 UIMC1-22 − AAUCCGCCCCGGAAUCGGGA 20 1301 UIMC1-23 + ACGCCGGGACCGGCCAUUAC 20 1302 UIMC1-24 − ACUUAACCAACCCCCGCCGC 20 1303 UIMC1-25 − AGCCCACUUCGGCGUCCGGA 20 1304 UIMC1-26 + AGGUAGGCCUCUCCCGACGC 20 1305 UIMC1-27 + CCCGAUUCCGGGGCGGAUUC 20 1306 UIMC1-28 + CCCGCGCAGUUGGUACGUCC 20 1307 UIMC1-29 + CCGCCCCGAGAGCGUGUCUC 20 1308 UIMC1-30 − CCGGAAUCCGCCCCGGAAUC 20 1309 UIMC1-31 − CCGGGACGUACCAACUGCGC 20 1310 UIMC1-32 + CGCAGUUGGUACGUCCCGGA 20 1311 UIMC1-33 + CGGCCCGCUACUCUCCGGGA 20 1312 UIMC1-34 + CGUCCCGGAUGGCUCCCCCG 20 1313 UIMC1-35 − UAAUGGCCGGUCCCGGCGUC 20 1314 UIMC1-36 − UCCGGAAUCCGCCCCGGAAU 20 1315 UIMC1-37 + UCCGGACGCCGAAGUGGGCU 20 1316 UIMC1-38 − UCCGGGACGUACCAACUGCG 20 1317 UIMC1-39 + UGUCUCUCGCGACGCGGGGG 20 1318 UIMC1-40 − UUAACCAACCCCCGCCGCGG 20 1319

Table II.40A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., UIMC1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the UIMC1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the UIMC1 gene.

TABLE II.40A Exemplary HDR-enhancing gRNAs Targeting a UIMC1 Gene A high level of orthogonality, starts with a G, PAM is NNGRRT S. aureus 40A 1st Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID UIMC1-41 − GCGUCGGGAAGCGCCCCUCCCG 22 1320 UIMC1-42 + GCUGGCCUUGCCGAAGUCGGGG 22 1321 UIMC1-43 + GUCUACAGAGCGGCCUGCGCCA 22 1322

Table II.40B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., UIMC1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the UIMC1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the UIMC1 gene.

TABLE II.40B Exemplary HDR-enhancing gRNAs Targeting a UIMC1 Gene A high level of orthogonality, and PAM is NNGRRT S. aureus 40B 2nd Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID UIMC1-44 + ACAGAGCGGCCUGCGCCAGCGA 22 1323 UIMC1-45 − AGUAAUGGCCGGUCCCGGCGUC 22 1324 UIMC1-46 − CAGGCCGCUCUGUAGACCUUCU 22 1325 UIMC1-47 + CCCUGCCUCCUUUUCUUCCUCA 22 1326 UIMC1-48 − CCGGAAUCCGCCCCGGAAUCGG 22 1327 UIMC1-49 + CCGGGACCGGCCAUUACUGGUG 22 1328 UIMC1-50 + CCGGGGCGGCCCUUCCUGAUGC 22 1329 UIMC1-51 + CCUCCCGCGCAGUUGGUACGUC 22 1330 UIMC1-52 + CCUCCGGACGCCGAAGUGGGCU 22 1331 UIMC1-53 + CGGGGCUGGCCUUGCCGAAGUC 22 1332 UIMC1-54 + CGGGGUGUGUCUCUCGCGACGC 22 1333 UIMC1-55 + CUGGGACCCUCCCGAUUCCGGG 22 1334 UIMC1-56 + UCCCGGAUGGCUCCCCCGCGGC 22 1335 UIMC1-57 − UCCCGGCGUCCGGAAUCCGCCC 22 1336 UIMC1-58 − UGAGGAAGAAAAGGAGGCAGGG 22 1337 UIMC1-59 − UGGGCGGAGCUGUGCGCAGGCG 22 1338 UIMC1-60 − UGUAGACCUUCUCCGGGUUGCC 22 1339

Table II.41A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RAD52 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RAD52 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RAD52 gene.

TABLE II.41A Exemplary HDR-enhancing gRNAs Targeting a RAD52 Gene A high level of orthogonality, and starts with a G S. pyogenes 41A 1st Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID RAD52-1 − GAACCCACGCCCAGCGCCGG 20 1340 RAD52-2 − GAACCGUAAAUCAAGUCGGA 20 1341 RAD52-3 − GAAGGAACCGUAAAUCAAGU 20 1342 RAD52-4 − GAAGGGUGCGCGAGCGUCUC 20 1343 RAD52-5 + GAGAGCGGCUUCCCCCGGGG 20 1344 RAD52-6 + GAGGAGAGCGGCUUCCCCCG 20 1345 RAD52-7 − GAGGCCGCGCAGAGGAGAAU 20 1346 RAD52-8 − GCACACAGGGAGCUCGAUCU 20 1347 RAD52-9 − GCAGCCCCAGGUUCUCGACC 20 1348 RAD52-10 + GCUGCCCGAGGCGCGUAAGU 20 1349 RAD52-11 + GCUUCCGGGUCGAGAACCUG 20 1350 RAD52-12 − GGAACCGUAAAUCAAGUCGG 20 1351 RAD52-13 + GGAGGAGAGCGGCUUCCCCC 20 1352 RAD52-14 − GGAGGCCGCGCAGAGGAGAA 20 1353 RAD52-15 − GGCAGCGCGCGGUGCACACA 20 1354 RAD52-16 + GGGAGGAGAGCGGCUUCCCC 20 1355 RAD52-17 − GGGCAGCGCGCGGUGCACAC 20 1356 RAD52-18 − GGGGAAGAGAUCUUAGAUGG 20 1357 RAD52-19 − GGUGAACUAGAACAGGCCUC 20 1358 RAD52-20 + GGUGGUUCUAGCCGUGGGUG 20 1359

Table II.41B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RAD52 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RAD52 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RAD52 gene.

TABLE II.41B Exemplary HDR-enhancing gRNAs Targeting a RAD52 Gene A high level of orthogonality S. pyogenes 41B 2nd Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID RAD52-21 − AACCGUAAAUCAAGUCGGAG 20 1360 RAD52-22 − ACCCACGGCUAGAACCACCC 20 1361 RAD52-23 + AGCUUCCGGGUCGAGAACCU 20 1362 RAD52-24 − AGGGAGCUCGAUCUAGGCUA 20 1363 RAD52-25 − CACGGCUAGAACCACCCCGG 20 1364 RAD52-26 + CAGCUUCCGGGUCGAGAACC 20 1365 RAD52-27 − CCACGGCUAGAACCACCCCG 20 1366 RAD52-28 − CCCACGGCUAGAACCACCCC 20 1367 RAD52-29 + CCCCGGGGUGGUUCUAGCCG 20 1368 RAD52-30 + CCCGGGGUGGUUCUAGCCGU 20 1369 RAD52-31 + CCGAGGCGCGUAAGUGGGGG 20 1370 RAD52-32 − CCUCCCCCACUUACGCGCCU 20 1371 RAD52-33 − CGCGAGCGUCUCUGGGAAGA 20 1372 RAD52-34 + CGCUGCCCGAGGCGCGUAAG 20 1373 RAD52-35 − CUCGAUCUAGGCUAUGGACA 20 1374 RAD52-36 − CUUAGAUGGAGGCCGCGCAG 20 1375 RAD52-37 − UACGCGCCUCGGGCAGCGCG 20 1376 RAD52-38 − UCCGAACCCACGCCCAGCGC 20 1377 RAD52-39 − UCGAUCUAGGCUAUGGACAA 20 1378 RAD52-40 + UGCCCGAGGCGCGUAAGUGG 20 1379

Table II.42A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RAD52 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RAD52 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RAD52 gene.

TABLE II.42A Exemplary HDR-enhancing gRNAs Targeting a RAD52 Gene A high level of orthogonality, starts with a G, PAM is NNGRRT S. aureus 42A 1st Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID RAD52-41 + GGAGCGUGGGAAGGCUCAGCUU 22 1380 RAD52-42 + GGGGGAGGAGAGCGGCUUCCCC 22 1381

Table II.42B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RAD52 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RAD52 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RAD52 gene.

TABLE II.42B Exemplary HDR-enhancing gRNAs Targeting a RAD52 Gene A high level of orthogonality, and PAM is NNGRRT S. aureus 42B 2nd Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID RAD52-43 − AGGCCGCGCAGAGGAGAAUGGG 22 1382 RAD52-44 − CAGCAUCUCUACGCUGAGACCU 22 1383 RAD52-45 + CUGAGGUCUCAGCGUAGAGAUG 22 1384 RAD52-46 − CUUAGAUGGAGGCCGCGCAGAG 22 1385 RAD52-47 + UUCCCCCGGGGUGGUUCUAGCC 22 1386 RAD52-48 + UUUUCCCCCUCCGGCGCUGGGC 22 1387

Table II.43A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., ERCC1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the ERCC1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the ERCC1 gene.

TABLE II.43A Exemplary HDR-enhancing gRNAs Targeting a ERCC1 Gene A high level of orthogonality, and starts with a G S. pyogenes 43A 1st Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID ERCC1-1 − GAAAGACUGCAGAGGGAUCG 20 1388 ERCC1-2 + GAGCCAAUAGAAUCCGGUGG 20 1389 ERCC1-3 + GCACGGACUCGCACAGGACC 20 1390 ERCC1-4 + GCCGGAAGUGCUGCGAGCCC 20 1391 ERCC1-5 + GCCGGACGAACGGAAGGCGG 20 1392 ERCC1-6 + GCCUCUAGCGCUGGGUGUUG 20 1393 ERCC1-7 + GCGCGUGGGGGGAAUAGGUG 20 1394 ERCC1-8 + GCGGGUGGAGAUUGGCGCCG 20 1395 ERCC1-9 + GCGUCCAGAUGCUAGCCUCG 20 1396 ERCC1-10 − GGAGAUCCCGGGAGAGCCGU 20 1397 ERCC1-11 + GGAGCCAAUAGAAUCCGGUG 20 1398 ERCC1-12 + GGCCGGACGAACGGAAGGCG 20 1399 ERCC1-13 + GGCGCUGAAACCGUGAGGCC 20 1400 ERCC1-14 + GGCGUCCAGAUGCUAGCCUC 20 1401 ERCC1-15 + GGCUUUGAAACUUAACAGUU 20 1402 ERCC1-16 − GGGAGAUCCCGGGAGAGCCG 20 1403 ERCC1-17 + GGGCCGGACGAACGGAAGGC 20 1404 ERCC1-18 + GGGCGUCCAGAUGCUAGCCU 20 1405 ERCC1-19 − GGUCCUGUGCGAGUCCGUGC 20 1406 ERCC1-20 + GUUACAGAGCCUCUAGCGCU 20 1407

Table II.43B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., ERCC1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the ERCC1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the ERCC1 gene.

TABLE II.43B Exemplary HDR-enhancing gRNAs Targeting a ERCC1 Gene A high level of orthogonality S. pyogenes 43B 2nd Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID ERCC1-21 + ACGGAGCCAAUAGAAUCCGG 20 1408 ERCC1-22 − AGAUCGCAGGAGAUCCAACU 20 1409 ERCC1-23 + AGCCUCGGGGGCCGGACGAA 20 1410 ERCC1-24 + AGCCUCUAGCGCUGGGUGUU 20 1411 ERCC1-25 + AUAGAAUCCGGUGGGGGCGA 20 1412 ERCC1-26 − CCGGAGCUUACGGUUCAGUA 20 1413 ERCC1-27 + CGGAGCCAAUAGAAUCCGGU 20 1414 ERCC1-28 + CGUCCAGAUGCUAGCCUCGG 20 1415 ERCC1-29 + CGUUACAGAGCCUCUAGCGC 20 1416 ERCC1-30 − CUACGUUCUCAUCCCGCAGC 20 1417 ERCC1-31 − CUCACGGUUUCAGCGCCGCG 20 1418 ERCC1-32 + CUCGCGGCGCUGAAACCGUG 20 1419 ERCC1-33 + UCACCAGCACGGACUCGCAC 20 1420 ERCC1-34 − UCCCCCGCCUUCCGUUCGUC 20 1421 ERCC1-35 − UCCGAGAGCUCCAUAGCGUC 20 1422 ERCC1-36 − UCGCCCCCACCGGAUUCUAU 20 1423 ERCC1-37 − UCGGCAAUGAUUGGCUUCCG 20 1424 ERCC1-38 + UCGGGGGCCGGACGAACGGA 20 1425 ERCC1-39 − UGGAGGACCGCGGAGGUCGU 20 1426 ERCC1-40 − UUCCGUUCGUCCGGCCCCCG 20 1427

Table II.44A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., ERCC1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the ERCC1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site TS S) of the ERCC1 gene.

TABLE II.44A Exemplary HDR-enhancing gRNAs Targeting a ERCC1 Gene A high level of orthogonality, starts with a G, PAM is NNGRRT S. aureus 44A 1st Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID ERCC1-41 + GAAACUGAAGCCAAGUCAAUGU 22 1428 ERCC1-42 + GAAGCCCUUCCGGACUCCGGGG 22 1429 ERCC1-43 + GACCCCCAUCCCACGGCUCUCC 22 1430 ERCC1-44 − GAUCCCGGGAGAGCCGUGGGAU 22 1431 ERCC1-45 + GCGCCGCGGAAGCCAAUCAUUG 22 1432 ERCC1-46 − GCUGACCCAGAAUGGGCAGGUC 22 1433 ERCC1-47 + GGAAUAGGUGUGGAAUAAAUGA 22 1434 ERCC1-48 + GGACCUGACGCUAUGGAGCUCU 22 1435 ERCC1-49 + GGGAAGAGAGGAAGCGCGUGGG 22 1436 ERCC1-50 + GGGAUGGUGGGGACGGAGCCAA 22 1437 ERCC1-51 + GGGGCCGGACGAACGGAAGGCG 22 1438 ERCC1-52 − GGGGGAGCGCCUGACUCAGCCC 22 1439 ERCC1-53 + GGGGGGAAUAGGUGUGGAAUAA 22 1440

Table II.44B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., ERCC1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the ERCC1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the ERCC1 gene.

TABLE II.44B Exemplary HDR-enhancing gRNAs Targeting a ERCC1 Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 44B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID ERCC1-54 + AAGCGCGUGGGGGGAAUAGGUG 22 1441 ERCC1-55 + ACAGGUGCGGGAGGCGGAGACU 22 1442 ERCC1-56 − AGACACGUUCCCAGUGCUGACC 22 1443 ERCC1-57 − AGCCUCAAGGGAAAGACUGCAG 22 1444 ERCC1-58 − AUCGCUCCGCCCCUCGCCCCCA 22 1445 ERCC1-59 − AUGGGAGAUCCCGGGAGAGCCG 22 1446 ERCC1-60 − CCUCUCUGGCCCCGCUCCCCAG 22 1447 ERCC1-61 + CGGAGUUUUGUGGGGGACGGCU 22 1448 ERCC1-62 − CUCAGUAAGGAGAGACUUAAGU 22 1449 ERCC1-63 + CUUACUGAGAGGAGGGACCAAG 22 1450 ERCC1-64 − CUUCCUCUCUUCCCGGUCCUGU 22 1451 ERCC1-65 + UCCCAUCCCAGACCUGCCCAUU 22 1452 ERCC1-66 + UCCGCGGUCCUCCAGAACCAUA 22 1453 ERCC1-67 + UCUGUUCUCCACUGAGCCCUGC 22 1454 ERCC1-68 + UGAAGCCAAGUCAAUGUCUGAG 22 1455 ERCC1-69 + UGGCGUUACAGAGCCUCUAGCG 22 1456 ERCC1-70 − UGGGAGGAGAGAGAUGUGGCCU 22 1457 ERCC1-71 + UGUGAGUGGGGGGUUCCUGCUG 22 1458 ERCC1-72 − UUACUGAGCGCUUCUGUGUGCC 22 1459 ERCC1-73 + UUGUGGGGGACGGCUGUGAGUG 22 1460

Table II.45A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., ERCC4 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the ERCC4 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the ERCC4 gene.

TABLE II.45A Exemplary HDR-enhancing gRNAs Targeting a ERCC4 Gene S. pyogenes A high level of orthogonality, and starts with a G 1st Tier 45A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID ERCC4-1 + GAAGAGCUUCCAUGGAGUCA 20 1461 ERCC4-2 − GACUCCAUGGAAGCUCUUCC 20 1462 ERCC4-3 − GAGAGCCGAGUCCGAGAGGA 20 1463 ERCC4-4 + GAUUGCCAUGGCGCCGCUGC 20 1464 ERCC4-5 − GCCAUGGCAAUCCGUCGAGC 20 1465 ERCC4-6 − GCCGACUCCUAGUGGAGAGU 20 1466 ERCC4-7 + GCCGGCUCGACGGAUUGCCA 20 1467 ERCC4-8 + GCCUACUCUCCACUAGGAGU 20 1468 ERCC4-9 + GCGACCCGGAAGAGCUUCCA 20 1469 ERCC4-10 + GCUGGAGUACGAGCGACAGC 20 1470 ERCC4-11 + GGAAGAGCUUCCAUGGAGUC 20 1471 ERCC4-12 + GGCUGCCGUCCUCUCGGACU 20 1472 ERCC4-13 + GGCUGCGUUCGGCUGCGACC 20 1473 ERCC4-14 + GUACGAGCGACAGCUGGUGC 20 1474 ERCC4-15 − GUACUCCAGCAGCGGCGCCA 20 1475

Table II.45B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., ERCC4 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the ERCC4 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the ERCC4 gene.

TABLE II.45B Exemplary HDR-enhancing gRNAs Targeting a ERCC4 Gene S. pyogenes A high level of orthogonality 2nd Tier 45B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID ERCC4-16 − AGCCGGCUGCCCUGACUCCA 20 1476 ERCC4-17 + AGUCAGGGCAGCCGGCUCGA 20 1477 ERCC4-18 + AGUUCGGCCUACUCUCCACU 20 1478 ERCC4-19 + CACUAGGAGUCGGCUUCCUU 20 1479 ERCC4-20 − CGAAGAGAGCCGAGUCCGAG 20 1480 ERCC4-21 − CGAAGGAAGCCGACUCCUAG 20 1481 ERCC4-22 + CGGCUCUCUUCGGUUGAGUU 20 1482 ERCC4-23 + CGGCUUCCUUCGGCUGCGUU 20 1483 ERCC4-24 + CUCUCGGACUCGGCUCUCUU 20 1484 ERCC4-25 + CUGGAACUGCUCGACACUGA 20 1485 ERCC4-26 − UCGCAGCCGAACGCAGCCGA 20 1486 ERCC4-27 + UCGGCUGGCUGCCGUCCUCU 20 1487 ERCC4-28 − UGACUCCAUGGAAGCUCUUC 20 1488 ERCC4-29 + UGGAACUGCUCGACACUGAC 20 1489 ERCC4-30 − UGUCGCUCGUACUCCAGCAG 20 1490 ERCC4-31 + UUCCAUGGAGUCAGGGCAGC 20 1491

Table II.46A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., ERCC4 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the ERCC4 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the ERCC4 gene.

TABLE II.46A Exemplary HDR-enhancing gRNA Targeting a ERCC4 Gene S. aureus A high level of orthogonality,starts with a G, PAM is NNGRRT 1st Tier 46A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID ERCC4-32 − GGCCGAACUCAACCGAAGAGAG 22 1492

Table II.46B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., ERCC4 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the ERCC4 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the ERCC4 gene.

TABLE II.46B Exemplary HDR-enhancing gRNAs Targeting a ERCC4 Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 46B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID ERCC4-33 − CCCUGACUCCAUGGAAGCUCUU 22 1493 ERCC4-34 − CGAAGGAAGCCGACUCCUAGUG 22 1494 ERCC4-35 + CGGAUUGCCAUGGCGCCGCUGC 22 1495 ERCC4-36 + CUGCGACCCGGAAGAGCUUCCA 22 1496 ERCC4-37 + UCUCGGACUCGGCUCUCUUCGG 22 1497 ERCC4-38 + UGAGUUCGGCCUACUCUCCACU 22 1498 ERCC4-39 + UGGAGUCAGGGCAGCCGGCUCG 22 1499

Table II.47A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., PARP1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the PARP1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the PARP1 gene.

TABLE II.47A Exemplary HDR-enhancing gRNAs Targeting a PARP1 Gene S. pyogenes A high level of orthogonality, and starts with a G 1st Tier 47A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID PARP1-1 − GAACCCGCGUCCACGGGGCG 20 1500 PARP1-2 − GAUUGCUGAUGCCUGGCCGC 20 1501 PARP1-3 + GCAGGGGGCGCGCGCGCCGC 20 1502 PARP1-4 − GCCCACGGAACCCGCGUCCA 20 1503 PARP1-5 − GCCCCCUGCCGGCCGGGGGG 20 1504 PARP1-6 + GCCGCUCAGGCGCCUGCGGC 20 1505 PARP1-7 + GCGCACGCGAGGCGGCGAGG 20 1506 PARP1-8 + GCGCCGCCGGCCCCGCCCCG 20 1507 PARP1-9 − GCGCGCCCCCUGCCGGCCGG 20 1508 PARP1-10 − GCGCGCGCCCCCUGCCGGCC 20 1509 PARP1-11 − GCGGCGCGCGCGCCCCCUGC 20 1510 PARP1-12 − GCGUGCGCUCACCCAGCCGC 20 1511 PARP1-13 − GGAACCCGCGUCCACGGGGC 20 1512 PARP1-14 + GGCAGCGUGUUUCUAGGUCG 20 1513 PARP1-15 + GGCCGGUGCGGCGUGUUCGG 20 1514 PARP1-16 + GGCGUGUUCGGUGGCGGCUC 20 1515 PARP1-17 + GGGAACGGCGGUGGCCGGUG 20 1516 PARP1-18 + GGGUUCCGUGGGCGUUCCCG 20 1517 PARP1-19 + GGUGGCCGGUGCGGCGUGUU 20 1518 PARP1-20 + GGUGGCGGCUCUGGCCGCUC 20 1519

Table II.47B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., PARP1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the PARP1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the PARP1 gene.

TABLE II.47B Exemplary HDR-enhancing gRNAs Targeting a PARP1 Gene S. pyogenes A high level of orthogonality 2nd Tier 47B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID PARP1-21 + AACUCCGCCCCCCGGCCGGC 20 1520 PARP1-22 + AAUCUAUCAGGGAACGGCGG 20 1521 PARP1-23 + ACUCCGCCCCCCGGCCGGCA 20 1522 PARP1-24 + AGCAAUCUAUCAGGGAACGG 20 1523 PARP1-25 − CCACGGAACCCGCGUCCACG 20 1524 PARP1-26 − CCCACGGAACCCGCGUCCAC 20 1525 PARP1-27 + CCCCGUGGACGCGGGUUCCG 20 1526 PARP1-28 + CCCGUGGACGCGGGUUCCGU 20 1527 PARP1-29 + CCGCUCAGGCGCCUGCGGCU 20 1528 PARP1-30 + CCGUGGGCGUUCCCGCGGCC 20 1529 PARP1-31 − CCUGAUAGAUUGCUGAUGCC 20 1530 PARP1-32 − CCUGGCCGCGGGAACGCCCA 20 1531 PARP1-33 + CGAGGCGGCAGCGUGUUUCU 20 1532 PARP1-34 − CGCCACCGAACACGCCGCAC 20 1533 PARP1-35 − CGGAACCCGCGUCCACGGGG 20 1534 PARP1-36 + CGGCUGGGUGAGCGCACGCG 20 1535 PARP1-37 + CGGUGCGGCGUGUUCGGUGG 20 1536 PARP1-38 + CUGGGUGAGCGCACGCGAGG 20 1537 PARP1-39 + UAUCAGGGAACGGCGGUGGC 20 1538 PARP1-40 + UGAGCGCACGCGAGGCGGCG 20 1539

Table II.48A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., PARP1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the PARP1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the PARP1 gene.

TABLE II.48A Exemplary HDR-enhancing gRNA Targeting a PARP1 Gene S. aureus A high level of orthogonality, starts with a G, PAM is NNGRRT 1st Tier 48A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID PARP1-41 − GCGCCCCCUGCCGGCCGGGGGG 22 1540

Table II.48B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., PARP1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the PARP1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the PARP1 gene.

TABLE II.48B Exemplary HDR-enhancing gRNAs Targeting a PARP1 Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 48B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID PARP1-42 + CCGCCGGCCCCGCCCCGUGGAC 22 1541 PARP1-43 + CUGGCCGCUCAGGCGCCUGCGG 22 1542

Table II.49A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., BRCA1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the BRCA1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the BRCA1 gene.

TABLE II.49A Exemplary HDR-enhancing gRNAs Targeting a BRCA1 Gene S. pyogenes A high level of orthogonality, and starts with a G 1st Tier 49A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID BRCA1-1 − GAAAGAGCCAAGCGUCUCUC 20 1543 BRCA1-2 + GAGGCCUUCACCCUCUGCUC 20 1544 BRCA1-3 + GAGUCCCGGGAAAGGGACAG 20 1545 BRCA1-4 + GAUGCUCUGGGGUACUGGCG 20 1546 BRCA1-5 + GCUCGCUGAGACUUCCUGGA 20 1547 BRCA1-6 + GCUGCUUAGCGGUAGCCCCU 20 1548 BRCA1-7 − GGGCCCCCUGUCCCUUUCCC 20 1549 BRCA1-8 + GGGGCCCAAGUGAUGCUCUG 20 1550 BRCA1-9 + GGGGGCCCAAGUGAUGCUCU 20 1551 BRCA1-10 + GGGGUACUGGCGUGGGAGAG 20 1552 BRCA1-11 + GGGUAAAGGUAGUAGAGUCC 20 1553 BRCA1-12 + GGUAAAGGUAGUAGAGUCCC 20 1554 BRCA1-13 − GGUACAAUCAGAGGAUGGGA 20 1555 BRCA1-14 + GGUAGUAGAGUCCCGGGAAA 20 1556 BRCA1-15 − GGUGAAGGCCUCCUGAGCGC 20 1557 BRCA1-16 − GUGAAGGCCUCCUGAGCGCA 20 1558 BRCA1-17 + GUGAGCUCGCUGAGACUUCC 20 1559 BRCA1-18 + GUGGGGUUUCUCAGAUAACU 20 1560

Table II.49B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., BRCA1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the BRCA1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the BRCA1 gene.

TABLE II.49B Exemplary HDR-enhancing gRNAs Targeting a BRCA1 Gene S. pyogenes A high level of orthogonality 2nd Tier 49B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID BRCA1-19 − AAAGAGCCAAGCGUCUCUCG 20 1561 BRCA1-20 + AAAUUAAAACUGCGACUGCG 20 1562 BRCA1-21 + ACGUCAUCCGGGGGCAGACU 20 1563 BRCA1-22 − CCAAGCGUCUCUCGGGGCUC 20 1564 BRCA1-23 + CCCCUUGGUUUCCGUGGCAA 20 1565 BRCA1-24 − CCCGCGCUUUUCCGUUGCCA 20 1566 BRCA1-25 + CCGUGGCAACGGAAAAGCGC 20 1567 BRCA1-26 − CCGUUGCCACGGAAACCAAG 20 1568 BRCA1-27 − CCUCUCAGAAUACGAAAUCA 20 1569 BRCA1-28 − CGAAAUCAAGGUACAAUCAG 20 1570 BRCA1-29 + CGGUAGCCCCUUGGUUUCCG 20 1571 BRCA1-30 − CUGCCCCCGGAUGACGUAAA 20 1572 BRCA1-31 + CUUUCCUUUUACGUCAUCCG 20 1573 BRCA1-32 + UACGUCAUCCGGGGGCAGAC 20 1574 BRCA1-33 + UCAUCCGGGGGCAGACUGGG 20 1575 BRCA1-34 + UCCGUGGCAACGGAAAAGCG 20 1576 BRCA1-35 − UCCGUUGCCACGGAAACCAA 20 1577 BRCA1-36 + UCUUUCCUUUUACGUCAUCC 20 1578 BRCA1-37 − UUCCGUUGCCACGGAAACCA 20 1579 BRCA1-38 + UUUCCUUUUACGUCAUCCGG 20 1580

Table II.50A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., BRCA1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the BRCA1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the BRCA1 gene.

TABLE II.50A Exemplary HDR-enhancing gRNAs Targeting a BRCA1 Gene S. aureus A high level of orthogonality, starts with a G, PAM is NNGRRT 1st Tier 50A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID BRCA1-39 − GAGCCAAGCGUCUCUCGGGGCU 22 1581 BRCA1-40 − GGAUUGGCCACCCAGUCUGCCC 22 1582

Table II.50B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., BRCA1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the BRCA1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the BRCA1 gene.

TABLE II.50B Exemplary HDR-enhancing gRNAs Targeting a BRCA1 Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 50B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID BRCA1-41 − AAAAGGAAAGAGACGGAAGAGG 22 1583 BRCA1-42 + ACAGGGGGCCCAAGUGAUGCUC 22 1584 BRCA1-43 − AUACGAAAUCAAGGUACAAUCA 22 1585 BRCA1-44 + AUGCUCUGGGGUACUGGCGUGG 22 1586 BRCA1-45 + CAGGAGGCCUUCACCCUCUGCU 22 1587 BRCA1-46 + CCUCUGCUCUGGGUAAAGGUAG 22 1588 BRCA1-47 − CUACCGCUAAGCAGCAGCCUCU 22 1589 BRCA1-48 − UCUACUACCUUUACCCAGAGCA 22 1590 BRCA1-49 + UCUGGGGUACUGGCGUGGGAGA 22 1591 BRCA1-50 + UUCCGUGGCAACGGAAAAGCGC 22 1592 BRCA1-51 + UUCCUGGACGGGGGACAGGCUG 22 1593 BRCA1-52 + UUUUACGUCAUCCGGGGGCAGA 22 1594

Table II.51A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RBBP8 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RBBP8 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RBBP8 gene.

TABLE II.51A Exemplary HDR-enhancing gRNAs Targeting a RBBP8 Gene S. pyogenes A high level of orthogonality, and starts with a G 1st Tier 51A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID RBBP8-1 + GAAUCCCGAGGCAAUCUCGG 20 1595 RBBP8-2 − GACAGCCCGCGCUUUAAGGC 20 1596 RBBP8-3 − GAGCCCGCGCGACGUCACGC 20 1597 RBBP8-4 − GAGGAGCGGGCUCUUCGGUG 20 1598 RBBP8-5 − GAUUCGCGAAAGCCCCCGAA 20 1599 RBBP8-6 + GCCAGACCCGCACGCGGAAC 20 1600 RBBP8-7 − GCCCGCGCCGGUUCCGCGUG 20 1601 RBBP8-8 + GCCCGGGCUACACUCGGUGG 20 1602 RBBP8-9 − GCCGGUUCCGCGUGCGGGUC 20 1603 RBBP8-10 + GCCUUAAAGCGCGGGCUGUC 20 1604 RBBP8-11 − GCUUUAAGGCCGGGGGCUGC 20 1605 RBBP8-12 − GGAGCCCGCGCGACGUCACG 20 1606 RBBP8-13 − GGAUUCGCGAAAGCCCCCGA 20 1607 RBBP8-14 + GGCGAAGGGCUCCCGGGGUA 20 1608 RBBP8-15 + GGCUCGCGCGCGCGCUUCGG 20 1609 RBBP8-16 − GGGGCGGGCUUGGCGGCGAA 20 1610 RBBP8-17 + GGGGGCUUUCGCGAAUCCCG 20 1611 RBBP8-18 − GGUAGCGCUCGUCCUCCCGC 20 1612 RBBP8-19 − GUCGCUCCGACCCAGAGCUC 20 1613 RBBP8-20 + GUGCUUGGCGAAGGGCUCCC 20 1614

Table II.51B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RBBP8 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RBBP8 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RBBP8 gene.

TABLE II.51B Exemplary HDR-enhancing gRNAs Targeting a RBBP8 Gene S. pyogenes A high level of orthogonality 2nd Tier 51B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID RBBP8-21 + AACUCCCGCGUGACGUCGCG 20 1615 RBBP8-22 + ACCCGCACGCGGAACCGGCG 20 1616 RBBP8-23 − ACCGCCUCCGAGAUUGCCUC 20 1617 RBBP8-24 + ACGAAGUGCGCCGCCGCGAU 20 1618 RBBP8-25 + ACGUCGCGCGGGCUCCCGGG 20 1619 RBBP8-26 + ACUCCCGCGUGACGUCGCGC 20 1620 RBBP8-27 − AGCCCGCGCUUUAAGGCCGG 20 1621 RBBP8-28 + CAACCAUCGCCCUCCGGGAU 20 1622 RBBP8-29 + CACGAAGUGCGCCGCCGCGA 20 1623 RBBP8-30 − CAUCCCGGAGGGCGAUGGUU 20 1624 RBBP8-31 − CCCGCGCCGGUUCCGCGUGC 20 1625 RBBP8-32 + CGCGAAUCCCGAGGCAAUCU 20 1626 RBBP8-33 + CGCGCGCUUCGGAGGUUUUU 20 1627 RBBP8-34 + CGGGCCCGGGCUACACUCGG 20 1628 RBBP8-35 + CGUGACGUCGCGCGGGCUCC 20 1629 RBBP8-36 + UAAAGCGCGGGCUGUCCGGA 20 1630 RBBP8-37 + UCCCGAGGCAAUCUCGGAGG 20 1631 RBBP8-38 − UCCGGACAGCCCGCGCUUUA 20 1632 RBBP8-39 − UCGGUGCGGCCCAUCCCGGA 20 1633 RBBP8-40 + UUAAAGCGCGGGCUGUCCGG 20 1634

Table II.52A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RBBP8 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RBBP8 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RBBP8 gene.

TABLE II.52A Exemplary HDR-enhancing gRNAs Targeting a RBBP8 Gene A high level of orthogonality, starts with a G, PAM is NNGRRT S. aureus 52A 1st Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID RBBP8-41 + GAGCGCUACCUCAGUACUACUU 22 1635 RBBP8-42 − GCAGUCGCUCCGACCCAGAGCU 22 1636 RBBP8-43 − GGGAGCCCGCGCGACGUCACGC 22 1637 RBBP8-44 + GGGUAGGGGUGGCUCCCGGCUC 22 1638 RBBP8-45 − GGUGCCCGCGCCGGUUCCGCGU 22 1639 RBBP8-46 + GUCGCGCGGGCUCCCGGGCGGG 22 1640

Table II.52B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RBBP8 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RBBP8 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RBBP8 gene.

TABLE II.52B Exemplary HDR-enhancing gRNAs Targeting a RBBP8 Gene A high level of orthogonality, and PAM is NNGRRT S. aureus 52B 2nd Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID RBBP8-47 + CCUUAAAGCGCGGGCUGUCCGG 22 1641 RBBP8-48 − CGCUCCGACCCAGAGCUCCGGG 22 1642 RBBP8-49 + CGGAGGGGUCGGCUUUCCCACC 22 1643 RBBP8-50 + CGGCGCGGGCACCUGGGGAGAA 22 1644 RBBP8-51 − CUCACCGCCUCCGAGAUUGCCU 22 1645 RBBP8-52 − CUCUUUCGCCCUUUUCCCUCAC 22 1646 RBBP8-53 + CUUGGCGAAGGGCUCCCGGGGU 22 1647 RBBP8-54 + UCCCGAGGCAAUCUCGGAGGCG 22 1648 RBBP8-55 − UCCUCCCGCCGGUCCACCACCA 22 1649 RBBP8-56 + UCGCUUCCCUUCGGGGGCUUUC 22 1650 RBBP8-57 + UCUCUUUACCCCACCCGGAGCU 22 1651 RBBP8-58 + UGCGUGCUUGGCGAAGGGCUCC 22 1652

Table II.53A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., EXO1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the EXO1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the EXO1 gene.

TABLE II.53A Exemplary HDR-enhancing gRNAs Targeting an EXO1 Gene A high level of orthogonality, and starts with a G S. pyogenes 53A 1st Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID EXO1-1 − GACGCGCAGGUCGACCCCCA 20 1653 EXO1-2 + GACGUCACAUCCUCUGGGCG 20 1654 EXO1-3 + GAGAGCAGACGAUUCCGGGC 20 1655 EXO1-4 + GAGGAGAGUCCCUUCUCGGA 20 1656 EXO1-5 − GAGGGUCGGAGGUGACGCGC 20 1657 EXO1-6 + GAUAAGAGAGCAGACGAUUC 20 1658 EXO1-7 + GCACAUCUCCGCGAGACAGA 20 1659 EXO1-8 + GCCUAAGGAAACGUGUCGUC 20 1660 EXO1-9 − GCGGAAAAAUGAGGUAAGUC 20 1661 EXO1-10 + GCGGGCUGUGCGGAGGCUAA 20 1662 EXO1-11 + GCGUUGACGUCACAUCCUCU 20 1663 EXO1-12 + GCUAAUGGGUGGGUUCCCUU 20 1664 EXO1-13 − GCUGACCUUUCAAUUUGCGC 20 1665 EXO1-14 + GGAAACGUGUCGUCUGGAAU 20 1666 EXO1-15 + GGCUAAUGGGUGGGUUCCCU 20 1667 EXO1-16 − GGCUGACCUUUCAAUUUGCG 20 1668 EXO1-17 + GGGAUUCGGGUCUUCCAGGA 20 1669 EXO1-18 + GUGAGUUAGGGGCGUCGGAG 20 1670 EXO1-19 + GUGUUCUGCGUUGCCGGCCG 20 1671 EXO1-20 + GUUGCCGGCCGUGGGUGCUC 20 1672

Table II.53B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., EXO1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the EXO1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the EXO1 gene.

TABLE II.53B Exemplary HDR-enhancing gRNAs Targeting an EXO1 Gene A high level of orthogonality S. pyogenes 53B 2nd Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID EXO1-21 + AAUCGGCUCCGCUCAAGGGG 20 1673 EXO1-22 + ACCGCAAUCGGCUCCGCUCA 20 1674 EXO1-23 − ACGCGCAGGUCGACCCCCAA 20 1675 EXO1-24 − ACGGCCGGCAACGCAGAACA 20 1676 EXO1-25 + AGGAACCCGCGCAAAUUGAA 20 1677 EXO1-26 + CAGCCUUUCGCGCGCUGUGU 20 1678 EXO1-27 + CCGACCCUCCUCUCGGGAUU 20 1679 EXO1-28 + CCGCAAUCGGCUCCGCUCAA 20 1680 EXO1-29 + CGCAAUCGGCUCCGCUCAAG 20 1681 EXO1-30 + CGCGUUGACGUCACAUCCUC 20 1682 EXO1-31 − CGGCCGGCAACGCAGAACAC 20 1683 EXO1-32 + CGGGUUUCUCCAACCGCAAU 20 1684 EXO1-33 − CUCGCGGAGAUGUGCAGGCG 20 1685 EXO1-34 − UCAACGCGUAUCCCGCAACC 20 1686 EXO1-35 + UCUCGGGAUUCGGGUCUUCC 20 1687 EXO1-36 − UGAGCGGAGCCGAUUGCGGU 20 1688 EXO1-37 − UGGAAGACCCGAAUCCCGAG 20 1689 EXO1-38 + UGUUCUGCGUUGCCGGCCGU 20 1690 EXO1-39 + UUACCCGUGUUCUGCGUUGC 20 1691 EXO1-40 − UUGCCUACACAGCGCGCGAA 20 1692

Table II.54A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., EXO1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the EXO1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the EXO1 gene.

TABLE II.54A Exemplary HDR-enhancing gRNAs Targeting an EXO1 Gene A high level of orthogonality, starts with a G, PAM is NNGRRT S. aureus 54A 1st Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID EXO1-41 + GAUUCCGGGCUGGAGCAGGCGC 22 1693 EXO1-42 − GCCUCCUGCGGCUUCCAACUCA 22 1694 EXO1-43 + GCGUCACCUCCGACCCUCCUCU 22 1695 EXO1-44 + GGAGAGCUCAGGACGCAACCCU 22 1696 EXO1-45 + GGAGCGGGCUGUGCGGAGGCUA 22 1697 EXO1-46 + GGCCGUGGGUGCUCUGGCCACA 22 1698 EXO1-47 + GGGUCUUCCAGGAAGGGAAGGA 22 1699 EXO1-48 − GGGUUCCUUGCGGCCCCGCCCA 22 1700

Table II.54B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., EXO1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the EXO1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the EXO1 gene.

TABLE II.54B Exemplary HDR-enhancing gRNAs Targeting an EXO1 Gene A high level of orthogonality, and PAM is NNGRRT S. aureus 54B 2nd Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID EXO1-49 − AAAGGCUGACCUUUCAAUUUGC 22 1701 EXO1-50 − AACCCUGGCGCCUGCUCCAGCC 22 1702 EXO1-51 + ACAGCGGAGCCCUUAGCCUGAG 22 1703 EXO1-52 + ACAGUGAGUUAGGGGCGUCGGA 22 1704 EXO1-53 + ACCCAACAGCGGAGCCCUUAGC 22 1705 EXO1-54 − ACUCAGGCUAAGGGCUCCGCUG 22 1706 EXO1-55 + AGGCCUAAGGAAACGUGUCGUC 22 1707 EXO1-56 + AGGCUAAUGGGUGGGUUCCCUU 22 1708 EXO1-57 − CCCACGGCCGGCAACGCAGAAC 22 1709 EXO1-58 + CCCGUGUUCUGCGUUGCCGGCC 22 1710 EXO1-59 + CCUCCGACCCUCCUCUCGGGAU 22 1711 EXO1-60 + CCUGCACAUCUCCGCGAGACAG 22 1712 EXO1-61 + CGCAGGAGGCGGAACCGGGUUG 22 1713 EXO1-62 + CGGCUCCGCUCAAGGGGAGGAG 22 1714 EXO1-63 + CGGGCUGUGCGGAGGCUAAUGG 22 1715 EXO1-64 − UCUCCUUCCCUUCCUGGAAGAC 22 1716 EXO1-65 − UGGAAGACCCGAAUCCCGAGAG 22 1717 EXO1-66 + UUGGAAGCCGCAGGAGGCGGAA 22 1718

Table II.55A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., DNA2 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the DNA2 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the DNA2 gene.

TABLE II.55A Exemplary HDR-enhancing gRNAs Targeting a DNA2 Gene A high level of orthogonality, and starts with a G S. pyogenes 55A 1st Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID DNA2-1 + GAACGAACUGGAGCUGCUGA 20 1719 DNA2-2 + GAAGAGUUUUUGGGAGGAGG 20 1720 DNA2-3 − GACAGAAAAGACAGCGGAAC 20 1721 DNA2-4 + GAGCCCUGCUGCUCAGGUGA 20 1722 DNA2-5 + GAUGGAGAAGAGUUUUUGGG 20 1723 DNA2-6 + GAUGGAGCAGCUGAACGAAC 20 1724 DNA2-7 − GCGCCGGCGCGUUCCACGUG 20 1725 DNA2-8 + GCGGCCUGGCGCAGGUCAUU 20 1726 DNA2-9 + GCUGAUGGAGAAGAGUUUUU 20 1727 DNA2-10 + GCUGCCGGCGGAGCUGUGAG 20 1728 DNA2-11 − GCUGCUCCAUCCUGGACGCG 20 1729 DNA2-12 + GGAACGCGCCGGCGCGGGAG 20 1730 DNA2-13 + GGAGAAGAGUUUUUGGGAGG 20 1731 DNA2-14 + GGGACAGAGCCCUGCUGCUC 20 1732 DNA2-15 + GGGAGGUUUCGGACACGGGU 20 1733 DNA2-16 + GGGCCCCACGUGGAACGCGC 20 1734 DNA2-17 − GGGCCCCUCACCUGAGCAGC 20 1735 DNA2-18 + GGUUGGAGUGUCAAGAGAGA 20 1736 DNA2-19 + GUAUUCCCAGUCCUAAGCAA 20 1737 DNA2-20 + GUGGAACGCGCCGGCGCGGG 20 1738

Table II.55B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., DNA2 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the DNA2 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the DNA2 gene.

TABLE II.55B Exemplary HDR-enhancing gRNAs Targeting a DNA2 Gene A high level of orthogonality S. pyogenes 55B 2nd Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID DNA2-21 + AACCCGGGAGGUUUCGGACA 20 1739 DNA2-22 − AACCCGUGUCCGAAACCUCC 20 1740 DNA2-23 − ACAGAAAAGACAGCGGAACC 20 1741 DNA2-24 + ACCCGGGAGGUUUCGGACAC 20 1742 DNA2-25 − ACCCGUGUCCGAAACCUCCC 20 1743 DNA2-26 − AGAAAAGACAGCGGAACCGG 20 1744 DNA2-27 + AGUCCUAAGCAAGGGAGCAA 20 1745 DNA2-28 + AGUUUGCGAUCCCCGCGUCC 20 1746 DNA2-29 + CACGUGGAACGCGCCGGCGC 20 1747 DNA2-30 − CAGAAAAGACAGCGGAACCG 20 1748 DNA2-31 + CCACGUGGAACGCGCCGGCG 20 1749 DNA2-32 − CCGCGCCGGCGCGUUCCACG 20 1750 DNA2-33 + CGCAUGCGCGCGAGGUGCGC 20 1751 DNA2-34 − CGCGCCGGCGCGUUCCACGU 20 1752 DNA2-35 + CGGCCUGGCGCAGGUCAUUU 20 1753 DNA2-36 + UGCGAUCCCCGCGUCCAGGA 20 1754 DNA2-37 + UGGAACGCGCCGGCGCGGGA 20 1755 DNA2-38 − UGUCCCAAAUGACCUGCGCC 20 1756 DNA2-39 − UUCGUUCAGCUGCUCCAUCC 20 1757 DNA2-40 − UUGCUCCCUUGCUUAGGACU 20 1758

Table II.56A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., DNA2 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the DNA2 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the DNA2 gene.

TABLE II.56A Exemplary HDR-enhancing gRNAs Targeting a DNA2 Gene A high level of orthogonality, starts with a G, PAM is NNGRRT S. aureus 56A 1st Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID DNA2-41 + GCGCAGGUCAUUUGGGACAUCU 22 1759 DNA2-42 + GUGAACCCGGGAGGUUUCGGAC 22 1760

Table II.56B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., DNA2 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the DNA2 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the DNA2 gene.

TABLE II.56B Exemplary HDR-enhancing gRNAs Targeting a DNA2 Gene A high level of orthogonality, and PAM is NNGRRT S. aureus 56B 2nd Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID DNA2-43 − AGACAGAAAAGACAGCGGAACC 22 1761 DNA2-44 + CCGGGAGGUUUCGGACACGGGU 22 1762 DNA2-45 + CGAACUGGAGCUGCUGAUGGAG 22 1763 DNA2-46 − CUUGCUUAGGACUGGGAAUACA 22 1764 DNA2-47 − CUUUGCUCCCUUGCUUAGGACU 22 1765 DNA2-48 + UACAGUUUGCGAUCCCCGCGUC 22 1766 DNA2-49 − UCAGCUGCUCCAUCCUGGACGC 22 1767 DNA2-50 − UCCAACCCGUGUCCGAAACCUC 22 1768

Table II.57A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., MRE11A gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the MRE11A gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the MRE11A gene.

TABLE II.57A Exemplary HDR-enhancing gRNAs Targeting a MRE11A Gene A high level of orthogonality, and starts with a G S. pyogenes 57A 1st Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID MRE11A-1 − GAACCCGGAAGUGAGAUGCA 20 1769 MRE11A-2 − GAGCCAAUCCUGAGCAGGCU 20 1770 MRE11A-3 + GAUUGGCUCCUGCGUGAGGG 20 1771 MRE11A-4 + GCCGCCUUGCAUCUCACUUC 20 1772 MRE11A-5 − GCCGUAAACCUGAAUUCCGC 20 1773 MRE11A-6 − GCGAGGCCCCGCCCUCACGC 20 1774 MRE11A-7 − GGCCGUAAACCUGAAUUCCG 20 1775 MRE11A-8 − GGCUACCGCACGCAGUGAGG 20 1776 MRE11A-9 − GGGCGGGGAAAGUAGCGGCG 20 1777 MRE11A-10 + GUAGCCAAUGAGAGCCGAAC 20 1778 MRE11A-11 + GUUCGUCUCCUAGCCUGCUC 20 1779 MRE11A-12 − GUUUCUCUCGCGACACUUCA 20 1780

Table II.57B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., MRE11A gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the MRE11A gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the MRE11A gene.

TABLE II.57B Exemplary HDR-enhancing gRNAs Targeting a MRE11A Gene A high level of orthogonality S. pyogenes 57B 2nd Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID MRE11A-13 − AAGUCCAGUUCGGCUCUCAU 20 1781 MRE11A-14 − ACCGCACGCAGUGAGGGGGC 20 1782 MRE11A-15 + ACGGACGCCGUUCUCUCCCG 20 1783 MRE11A-16 − AUUGGCUACCGCACGCAGUG 20 1784 MRE11A-17 + AUUGGCUCCUGCGUGAGGGC 20 1785 MRE11A-18 + CAGGAUUGGCUCCUGCGUGA 20 1786 MRE11A-19 + CCCCGCCCCCUCACUGCGUG 20 1787 MRE11A-20 − CCGCACGCAGUGAGGGGGCG 20 1788 MRE11A-21 + CCGCCUUGCAUCUCACUUCC 20 1789 MRE11A-22 − CGCAGGAGCCAAUCCUGAGC 20 1790 MRE11A-23 + CGUUCUCUCCCGCGGAAUUC 20 1791 MRE11A-24 − CUGAAUUCCGCGGGAGAGAA 20 1792 MRE11A-25 − UACCGCACGCAGUGAGGGGG 20 1793 MRE11A-26 − UAGAUGCUUCAAGUCCAGUU 20 1794 MRE11A-27 + UCAGGAUUGGCUCCUGCGUG 20 1795 MRE11A-28 + UCCCGCGGAAUUCAGGUUUA 20 1796 MRE11A-29 + UCUCCUAGCCUGCUCAGGAU 20 1797 MRE11A-30 − UGGCUACCGCACGCAGUGAG 20 1798 MRE11A-31 − UUGGCUACCGCACGCAGUGA 20 1799 MRE11A-32 + UUGGCUCCUGCGUGAGGGCG 20 1800

Table II.58A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., MRE11A gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the MRE11A gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site TS S) of the MRE11A gene.

TABLE II.58A Exemplary HDR-enhancing gRNA Targeting a MRE11A Gene A high level of orthogonality, starts with a G, PAM is NNGRRT S. aureus 58A 1st Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID MRE11A-33 − GAGAACCCGCAGGGCCGUAAAC 22 1801

Table II.58B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., MRE11A gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the MRE1UA gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site SAS) of the MRE11A gene.

TABLE II.58B Exemplary HDR-enhancing gRNAs Targeting a MRE11A Gene A high level of orthogonality, and PAM is NNGRRT S. aureus 58B 2nd Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID MRE11A-34 + AAACGGACGCCGUUCUCUCCCG 22 1802 MRE11A-35 + AUCGCCGCCUUGCAUCUCACUU 22 1803 MRE11A-36 + CAGACCGUGUUGUUUUCUUUUC 22 1804 MRE11A-37 + CGGAAUUCAGGUUUACGGCCCU 22 1805 MRE11A-38 + CGGGUUCGUCUCCUAGCCUGCU 22 1806 MRE11A-39 − UCCGUUUCUCUCGCGACACUUC 22 1807

Table II.59A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RAD50 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RAD50 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RAD50 gene.

TABLE II.59A Exemplary HDR-enhancing gRNAs Targeting a RAD50 Gene A high level of orthogonality, and starts with a G S. pyogenes 59A 1st Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID RAD50-1 − GAAGCAGAGGGCUAGGUGCU 20 1808 RAD50-2 + GAGAGCGGCGUGGACGCGUG 20 1809 RAD50-3 − GCAGCUCCGACUUCCGGGUG 20 1810 RAD50-4 − GCCGCUCUCCUGGGGCACGC 20 1811 RAD50-5 − GCCGGGAAAUCAGAGUCCCG 20 1812 RAD50-6 + GCCGUACCGCACCCGGAAGU 20 1813 RAD50-7 + GCGGGGUCGCAUUGUGGCUA 20 1814 RAD50-8 + GCGGUUGCGGGGUCGCAUUG 20 1815 RAD50-9 − GCGUCCACGCCGCUCUCCUG 20 1816 RAD50-10 − GCGUGCGCAGCUCCGACUUC 20 1817 RAD50-11 + GCUGUGAGUGCGCGGUUGCG 20 1818 RAD50-12 + GGCAGGAAGCUGUGAGUGCG 20 1819 RAD50-13 + GGCCCACGUGAUCCGCAGGG 20 1820 RAD50-14 − GGCCGCCCUGCGGAUCACGU 20 1821 RAD50-15 − GGUGCGGUACGGCGAAGCAG 20 1822 RAD50-16 − GGUGCUGGGUGCUGUUGCCA 20 1823 RAD50-17 − GGUGCUGUUGCCAGGGGCAG 20 1824 RAD50-18 − GUACGGCGAAGCAGAGGGCU 20 1825 RAD50-19 − GUGCGGUACGGCGAAGCAGA 20 1826 RAD50-20 + GUGGACGCGUGCGGGCCUAG 20 1827

Table II.59B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RAD50 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RAD50 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RAD50 gene.

TABLE II.59B Exemplary HDR-enhancing gRNAs Targeting a RAD50 Gene S. pyogenes A high level of orthogonality 2nd Tier 59B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID RAD50-21 + AAGCUGUGAGUGCGCGGUUG 20 1828 RAD50-22 + ACCGCGGGACUCUGAUUUCC 20 1829 RAD50-23 − ACGCGUCCACGCCGCUCUCC 20 1830 RAD50-24 + AGAGCGGCGUGGACGCGUGC 20 1831 RAD50-25 + AGAGGCCCACGUGAUCCGCA 20 1832 RAD50-26 + AGCUGUGAGUGCGCGGUUGC 20 1833 RAD50-27 + AUCCGCAGGGCGGCCGAGGC 20 1834 RAD50-28 + CCCGGCGUGCCCCAGGAGAG 20 1835 RAD50-29 − CCGCUCUCCUGGGGCACGCC 20 1836 RAD50-30 − CGAAGCAGAGGGCUAGGUGC 20 1837 RAD50-31 + CGGAGCUGCGCACGCACCGC 20 1838 RAD50-32 − CGGCCGCCCUGCGGAUCACG 20 1839 RAD50-33 + CGUGAUCCGCAGGGCGGCCG 20 1840 RAD50-34 − CGUGCGCAGCUCCGACUUCC 20 1841 RAD50-35 + CUGAUUUCCCGGCGUGCCCC 20 1842 RAD50-36 + CUGCUUCGCCGUACCGCACC 20 1843 RAD50-37 + UAGAGGCCCACGUGAUCCGC 20 1844 RAD50-38 − UCCGACUUCCGGGUGCGGUA 20 1845 RAD50-39 + UCGGAGCUGCGCACGCACCG 20 1846 RAD50-40 − UGCGGAUCACGUGGGCCUCU 20 1847

Table II.60A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RAD50 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RAD50 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RAD50 gene.

TABLE II.60A Exemplary HDR-enhancing gRNAs Targeting a RAD50 Gene S. aureus A high level of orthogonality, starts with a G, PAM is NNGRRT 1st Tier 60A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID RAD50-41 + GGAAGCUGUGAGUGCGCGGUUG 22 1848 RAD50-42 − GGUGCGUGCGCAGCUCCGACUU 22 1849

Table II.60B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., RAD50 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the RAD50 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the RAD50 gene.

TABLE II.60B Exemplary HDR-enhancing gRNAs Targeting a RAD50 Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 60B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID RAD50-43 − AGCUUCCUGCCUCGGCCGCCCU 22 1850 RAD50-44 + AGGGCGGCCGAGGCAGGAAGCU 22 1851 RAD50-45 − CGGCGAAGCAGAGGGCUAGGUG 22 1852 RAD50-46 − CUCCUGGGGCACGCCGGGAAAU 22 1853

Table II.61A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., NBN gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the NBN gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the NBN gene.

TABLE II.61A Exemplary HDR-enhancing gRNAs Targeting a NBN Gene S. pyogenes A high level of orthogonality, and starts with a G 1st Tier 61A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID NBN-1 − GAGCGCGGAUACGGCGCCUG 20 1854 NBN-2 − GAUGAGGCGGGAGUGCGACU 20 1855 NBN-3 − GCAGGCUGCCUUGGAUGAGG 20 1856 NBN-4 − GCGGAUACGGCGCCUGCGGU 20 1857 NBN-5 − GGCGCUUGCCCGCCACCUGG 20 1858 NBN-6 − GGGAGCCACGCAGGCUGCCU 20 1859 NBN-7 − GUUAAAAGGGUAUGUUUCUA 20 1860

Table II.61B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., NBN gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the NBN gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the NBN gene.

TABLE II.61B Exemplary HDR-enhancing gRNAs Targeting a NBN Gene S. pyogenes A high level of orthogonality 2nd Tier 61B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID NBN-8 − ACGGCGCCUGCGGUCGGCAU 20 1861 NBN-9 − AUGAGGCGGGAGUGCGACUU 20 1862 NBN-10 − CACGCAGGCUGCCUUGGAUG 20 1863 NBN-11 − CAGGCUGCCUUGGAUGAGGC 20 1864 NBN-12 + CCGGAGCCCAUGCCGACCGC 20 1865 NBN-13 − CCUGCGGUCGGCAUGGGCUC 20 1866 NBN-14 − CUAAGGUGUCGCUGAAUGUA 20 1867 NBN-15 + CUCAUCCAAGGCAGCCUGCG 20 1868 NBN-16 − CUGCGGUCGGCAUGGGCUCC 20 1869 NBN-17 − CUGCUAGACGAGCGCGGAUA 20 1870 NBN-18 + CUGUUCCUUUUCCAACCACC 20 1871 NBN-19 − CUUGCCCGCCACCUGGUGGU 20 1872 NBN-20 + CUUUUCCAACCACCAGGUGG 20 1873 NBN-21 − UACGGCGCCUGCGGUCGGCA 20 1874 NBN-22 + UCCCGGGAGCGCGCACGUCC 20 1875 NBN-23 − UCCGGGACGUGCGCGCUCCC 20 1876 NBN-24 + UCGCACUCCCGCCUCAUCCA 20 1877 NBN-25 − UGAAAUGUGCUGCGUUAAAA 20 1878 NBN-26 − UUGAAAUGUGCUGCGUUAAA 20 1879 NBN-27 − UUGGGCGCUUGCCCGCCACC 20 1880

Table II.62A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., NBN gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the NBN gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the NBN gene.

TABLE II.62A Exemplary HDR-enhancing gRNA Targeting a NBN Gene S. aureus A high level oforthogonality, starts with a G, PAM is NNGRRT 1st Tier 62A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID NBN-28 − GUAUUGAAAUGUGCUGCGUUAA 22 1881

Table II.62B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., NBN gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the NBN gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the NBN gene.

TABLE II.62B Exemplary HDR-enhancing gRNAs Targeting a NBN Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 62B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID NBN-29 − AGGGUAUGUUUCUAAGGUGUCG 22 1882 NBN-30 − CCCGGGAGCCACGCAGGCUGCC 22 1883 NBN-31 − CGCAGGCUGCCUUGGAUGAGGC 22 1884

Table II.63A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., MSH2 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the MSH2 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the MSH2 gene.

TABLE II.63A Exemplary HDR-enhancing gRNAs Targeting a MSH2 Gene S. pyogenes A high level of orthogonality, and starts with a G 1st Tier 63A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID MSH2-1 + GAAACAGCUUAGUGGGUGUG 20 1885 MSH2-2 + GAGGCGGGAAACAGCUUAGU 20 1886 MSH2-3 + GCAGCUGAGUAAACACAGAA 20 1887 MSH2-4 − GCCCAGCUUCCCGCGCACGC 20 1888 MSH2-5 + GCCGCUCGGGGGACGUGGGA 20 1889 MSH2-6 + GCCGUGGCCGGACGCCGCUC 20 1890 MSH2-7 + GCUAAAGUCACCAGCGUGCG 20 1891 MSH2-8 − GCUGCAAGGCUUGAAGCCCC 20 1892 MSH2-9 + GGAAACAGCUUAGUGGGUGU 20 1893 MSH2-10 + GGACGCCGCUCGGGGGACGU 20 1894 MSH2-11 + GGAGGCGGGAAACAGCUUAG 20 1895 MSH2-12 + GGGAAACAGCUUAGUGGGUG 20 1896 MSH2-13 + GGGCCGCGUCUGCUUAUGAU 20 1897 MSH2-14 + GGGGACGUGGGAGGGGAGGC 20 1898 MSH2-15 + GGGGGACGUGGGAGGGGAGG 20 1899 MSH2-16 − GGUGGGGUGUAUGCAAGGGU 20 1900

Table II.63B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., MSH2 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the MSH2 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the MSH2 gene.

TABLE II.63B Exemplary HDR-enhancing gRNAs Targeting a MSH2 Gene S. pyogenes A high level of orthogonality 2nd Tier 63B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID MSH2-17 + ACCAGCGUGCGCGGGAAGCU 20 1901 MSH2-18 − AGGGCUGCGUUUCGGUGGGU 20 1902 MSH2-19 − AUGCCUGCGCCUAGGUCGCG 20 1903 MSH2-20 − CAACCAAUCAUAAGCAGACG 20 1904 MSH2-21 − CACGUCCCCCGAGCGGCGUC 20 1905 MSH2-22 − CAGGGCUGCGUUUCGGUGGG 20 1906 MSH2-23 − CCCCGAGCGGCGUCCGGCCA 20 1907 MSH2-24 + CCGCUCGGGGGACGUGGGAG 20 1908 MSH2-25 + CCGUGGCCGGACGCCGCUCG 20 1909 MSH2-26 + CGCCGUGGCCGGACGCCGCU 20 1910 MSH2-27 + CGUGGCCGGACGCCGCUCGG 20 1911 MSH2-28 + CUAAAGUCACCAGCGUGCGC 20 1912 MSH2-29 + CUACUAAGGAUGCGCGUCUG 20 1913 MSH2-30 + CUGAUUGGGUGUGGUCGCCG 20 1914 MSH2-31 + CUGCUUAUGAUUGGUUGCCG 20 1915 MSH2-32 + UACUAAGGAUGCGCGUCUGC 20 1916 MSH2-33 − UACUGCGCAUGCCUGCGCCU 20 1917 MSH2-34 + UGCGGGUUUCCGCGCGACCU 20 1918 MSH2-35 + UUGGGUGUGGUCGCCGUGGC 20 1919 MSH2-36 + UUUCCGCGCGACCUAGGCGC 20 1920

Table II.64A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., MSH2 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the MSH2 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the MSH2 gene.

TABLE II.64A Exemplary HDR-enhancing gRNAs Targeting a MSH2 Gene S. aureus A high level of orthogonality, starts with a G, PAM is NNGRRT 1st Tier 64A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID MSH2-37 + GAAACGCAGCCCUGGAAGCUGA 22 1921 MSH2-38 + GCGGGAAACAGCUUAGUGGGUG 22 1922 MSH2-39 + GCUCUACUAAGGAUGCGCGUCU 22 1923

Table II.64B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., MSH2 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the MSH2 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the MSH2 gene.

TABLE II.64B Exemplary HDR-enhancing gRNAs Targeting a MSH2 Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 64B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID MSH2-40 + AAACACAGAAAGGAGCUCUACU 22 1924 MSH2-41 − AGCCCCUGGGUGGGGUGUAUGC 22 1925 MSH2-42 + AGGGGAGGCGGGAAACAGCUUA 22 1926 MSH2-43 − CAGCUUCCAGGGCUGCGUUUCG 22 1927 MSH2-44 − CCAGGGCUGCGUUUCGGUGGGU 22 1928 MSH2-45 + CCAGGGGCUUCAAGCCUUGCAG 22 1929 MSH2-46 − UCAGCUGCAAGGCUUGAAGCCC 22 1930 MSH2-47 − UGCAAGGCUUGAAGCCCCUGGG 22 1931

Table II.65A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., MSH3 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the MSH3 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the MSH3 gene.

TABLE II.65A Exemplary HDR-enhancing gRNAs Targeting a MSH3 Gene S. pyogenes A high level of orthogonality, and starts with a G 1st Tier 65A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID MSH3-1 − GACCCUGCGUGCGCCGGGGC 20 1932 MSH3-2 − GAGACAUGGCAGGGCAAGGA 20 1933 MSH3-3 + GAGGCCCCGCCCCCCCGCCC 20 1934 MSH3-4 − GCAGGGCAAGGAUGGCAGCC 20 1935 MSH3-5 − GCCGCGACCCUGCGUGCGCC 20 1936 MSH3-6 − GCCUGCACAAAUGGGGACGA 20 1937 MSH3-7 + GCGGGCUCGCGCUCCUCGCC 20 1938 MSH3-8 + GCUCGCGCCCGCAGACGCCU 20 1939 MSH3-9 − GCUUCCGGCGAGACAUGGCA 20 1940 MSH3-10 − GGCAGCCCGGCGGCAGGGCC 20 1941 MSH3-11 − GGCUUCCGGCGAGACAUGGC 20 1942 MSH3-12 − GGGCAAGGAUGGCAGCCCGG 20 1943 MSH3-13 − GGGCCUCGCCUGCACAAAUG 20 1944 MSH3-14 − GGGGCCUCGCCUGCACAAAU 20 1945 MSH3-15 + GUCUCGCCGGAAGCCUGCGU 20 1946 MSH3-16 − GUGCGCCGGGGCGGGGGGGC 20 1947

Table II.65B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., MSH3 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the MSH3 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the MSH3 gene.

TABLE II.65B Exemplary HDR-enhancing gRNAs Targeting a MSH3 Gene S. pyogenes A high level of orthogonality 2nd Tier 65B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID MSH3-17 − AAGGAUGGCAGCCCGGCGGC 20 1948 MSH3-18 + ACGCCUGGGAACUGCGGCCG 20 1949 MSH3-19 − AGGAUGGCAGCCCGGCGGCA 20 1950 MSH3-20 − CAAAUGGGGACGAGGGGGGC 20 1951 MSH3-21 + CCCCGGCGCACGCAGGGUCG 20 1952 MSH3-22 + CCGCAGACGCCUGGGAACUG 20 1953 MSH3-23 − CCGCAGUUCCCAGGCGUCUG 20 1954 MSH3-24 − CCGCGACCCUGCGUGCGCCG 20 1955 MSH3-25 − CCUGCACAAAUGGGGACGAG 20 1956 MSH3-26 − CGACCCUGCGUGCGCCGGGG 20 1957 MSH3-27 − CGCAGGCUUCCGGCGAGACA 20 1958 MSH3-28 − CGCAGUUCCCAGGCGUCUGC 20 1959 MSH3-29 − CGCCGCGACCCUGCGUGCGC 20 1960 MSH3-30 − CGCCUGCACAAAUGGGGACG 20 1961 MSH3-31 + CGCCUGGGAACUGCGGCCGC 20 1962 MSH3-32 + CGUCCCCAUUUGUGCAGGCG 20 1963 MSH3-33 + CUUGCCCUGCCAUGUCUCGC 20 1964 MSH3-34 + UCAAGUUUGGCGCGAAAUUG 20 1965 MSH3-35 + UCUCGCCGGAAGCCUGCGUC 20 1966 MSH3-36 − UGGCGAGGAGCGCGAGCCCG 20 1967

Table II.66 provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., MSH3 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the MSH3 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the MSH3 gene.

TABLE II.66 Exemplary HDR-enhancing gRNAs Targeting a MSH3 Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 66B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID MSH3-37 + CGCCCCCCCGCCCCGGCGCACG 22 1968 MSH3-38 − UUCCGGCGAGACAUGGCAGGGC 22 1969

Table II.67A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., MSH6 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the MSH6 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the MSH6 gene.

TABLE II.67A Exemplary HDR-enhancing gRNAs Targeting a MSH6 Gene S. pyogenes A high level of orthogonality, and starts with a G 1st Tier 67A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID MSH6-1 − GACGUGGGGAAGGGCGGGGC 20 1970 MSH6-2 + GCGCCUGUUGAUUGGCCACU 20 1971 MSH6-3 − GCGCGCGGCGACGUGGGGAA 20 1972 MSH6-4 − GCGCUCCGCCGGAGGAACCC 20 1973 MSH6-5 − GCGGCGACGUGGGGAAGGGC 20 1974 MSH6-6 − GCUCCUGCUGGCGGGAAAUC 20 1975 MSH6-7 − GCUGGCACACUGGUGGGUAG 20 1976 MSH6-8 − GGCACACUGGUGGGUAGGGG 20 1977 MSH6-9 − GGCCCCAGUGGCCAAUCAAC 20 1978 MSH6-10 − GGCGCCUCGCCGUGCGCGGG 20 1979 MSH6-11 + GGCGCCUGUUGAUUGGCCAC 20 1980 MSH6-12 − GGCGGGGCUGGCACACUGGU 20 1981 MSH6-13 − GGCUGGCACACUGGUGGGUA 20 1982 MSH6-14 − GGCUGGCACGCUGGCGGUGA 20 1983 MSH6-15 − GGGCUGGCACACUGGUGGGU 20 1984 MSH6-16 − GGGCUGGCACGCUGGCGGUG 20 1985 MSH6-17 − GGGGAGGCGCGCUCCGCCGG 20 1986 MSH6-18 + GUCGCCGCGCGCCCGGGGGC 20 1987 MSH6-19 − GUGCGCGGGCGGUGCGCGCC 20 1988 MSH6-20 + GUUGAUUGGCCACUGGGGCC 20 1989

Table II.67B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., MSH6 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the MSH6 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the MSH6 gene.

TABLE II.67B Exemplary HDR-enhancing gRNAs Targeting a MSH6 Gene S. pyogenes A high level of orthogonality 2nd Tier 67B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID MSH6-21 − AAAGCACCGCAUCUACCGCG 20 1990 MSH6-22 − AACAGGCGCCUCGCCGUGCG 20 1991 MSH6-23 − ACAGGCGCCUCGCCGUGCGC 20 1992 MSH6-24 − ACCGCGCGGCUCCUGCUGGC 20 1993 MSH6-25 + ACGGCGAGGCGCCUGUUGAU 20 1994 MSH6-26 − AUCUACCGCGCGGCUCCUGC 20 1995 MSH6-27 + CAGGAGCCGCGCGGUAGAUG 20 1996 MSH6-28 + CCCCCAGAUUUCCCGCCAGC 20 1997 MSH6-29 − CCCCCGGGCGCGCGGCGACG 20 1998 MSH6-30 − CCCCGGGCGCGCGGCGACGU 20 1999 MSH6-31 + CGCACCGCCCGCGCACGGCG 20 2000 MSH6-32 + CGCCUGUUGAUUGGCCACUG 20 2001 MSH6-33 − CGCGCUCCGCCGGAGGAACC 20 2002 MSH6-34 − CGCGGCGACGUGGGGAAGGG 20 2003 MSH6-35 − CGGAGGAACCCGGGCCCCAG 20 2004 MSH6-36 + CGUCGCCGCGCGCCCGGGGG 20 2005 MSH6-37 − CUGGCGGGAAAUCUGGGGGG 20 2006 MSH6-38 − UACCGCGCGGCUCCUGCUGG 20 2007 MSH6-39 + UGGCGCGCACCGCCCGCGCA 20 2008 MSH6-40 + UGGGGCCCGGGUUCCUCCGG 20 2009

Table II.68A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., MSH6 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the MSH6 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the MSH6 gene.

TABLE II.68A Exemplary HDR-enhancing gRNA Targeting a MSH6 Gene S. aureus A high level of orthogonality, starts with a G, PAM is NNGRRT 1st Tier 68A gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID MSH6-41 − GAAGGGCGGGGCUGGCACACUG 22 2010

Table II.68B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., MSH6 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the MSH6 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the MSH6 gene.

TABLE II.68B Exemplary HDR-enhancing gRNAs Targeting a MSH6 Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 68B gRNA Target Site Seq Name DNA Strand Targeting Domain Length ID MSH6-42 + CCUGUUGAUUGGCCACUGGGGC 22 2011

Table II.69A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., MlH1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the MlH1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the MlH1 gene.

TABLE II.69A Exemplary HDR-enhancing gRNAs Targeting a MlH1 Gene S. pyogenes A high level of orthogonality, and starts with a G 1st Tier 69A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID MLH1-1 + GACAGUGGUGAACCGCAUCG 20 2012 MLH1-2 − GACUGGCACGUCAGGGAACC 20 2013 MLH1-3 + GCACGAGGCACUGAGGUGAU 20 2014 MLH1-4 + GCCAAAAUGUCGUUCGUGGC 20 2015 MLH1-5 + GCGCAAGCGCAUAUCCUUCU 20 2016 MLH1-6 + GCGCAUGCCCACAACGGCGG 20 2017 MLH1-7 − GCGCUGUACAUGCCUCUGCC 20 2018 MLH1-8 + GCGGACACGCCUCUUUGCCC 20 2019 MLH1-9 − GCUACUGCCCGCUACCUAGA 20 2020 MLH1-10 + GCUCCUAAAAACGAACCAAU 20 2021 MLH1-11 − GGAAACGUCUAGAUGCUCAA 20 2022 MLH1-12 + GGCAGGGGUUAUUCGGCGGC 20 2023 MLH1-13 + GGCCGCGUCACUCAAUGGCG 20 2024 MLH1-14 + GGUACGGAGGGAGUCGAGCC 20 2025 MLH1-15 + GGUGAACCGCAUCGCGGCGG 20 2026 MLH1-16 + GGUUCCCUGACGUGCCAGUC 20 2027 MLH1-17 − GGUUCGUUUUUAGGAGCUCG 20 2028 MLH1-18 − GUCCGCGCCAUUGAGUGACG 20 2029 MLH1-19 + GUCGAGCCGGGCUCACUUAA 20 2030 MLH1-20 + GUGGUGAACCGCAUCGCGGC 20 2031

Table II.69B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., MlH1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the MlH1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the MlH1 gene.

TABLE II.69B Exemplary HDR-enhancing gRNAs Targeting a MlH1 Gene S. pyogenes A high level of orthogonality 2nd Tier 69B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID MLH1-21 + ACAGCGCAUGCCCACAACGG 20 2032 MLH1-22 + ACUUAAGGGCUACGACUUAA 20 2033 MLH1-23 + AGUCGAGCCGGGCUCACUUA 20 2034 MLH1-24 + AGUGGUGAACCGCAUCGCGG 20 2035 MLH1-25 + AUGGCGUAAGCUACAGCUGA 20 2036 MLH1-26 + CCACAACGGCGGAGGCCGCC 20 2037 MLH1-27 + CCCACAACGGCGGAGGCCGC 20 2038 MLH1-28 − CCGGCGGCCUCCGCCGUUGU 20 2039 MLH1-29 + CGCAUAUCCUUCUAGGUAGC 20 2040 MLH1-30 + CGCGGACACGCCUCUUUGCC 20 2041 MLH1-31 + CGGCGGGGGAAGUUAUCCAG 20 2042 MLH1-32 + CGUUCGUGGCAGGGGUUAUU 20 2043 MLH1-33 + CUUAAGGGCUACGACUUAAC 20 2044 MLH1-34 + UAACGGGCCGCGUCACUCAA 20 2045 MLH1-35 − UAACUUCCCCCGCCGCGAUG 20 2046 MLH1-36 + UAGCGGGCAGUAGCCGCUUC 20 2047 MLH1-37 + UCGUGGCAGGGGUUAUUCGG 20 2048 MLH1-38 − UGAUAGCAUUAGCUGGCCGC 20 2049 MLH1-39 + UGGCGCCAAAAUGUCGUUCG 20 2050 MLH1-40 + UGGUGAACCGCAUCGCGGCG 20 2051

Table II.70A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., MlH1 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the MlH1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the MlH1 gene.

TABLE II.70A Exemplary HDR-enhancing gRNAs Targeting a MlH1 Gene S. aureus A high level of orthogonality, starts with a G, PAM is NNGRRT 1st Tier 70A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID MLH1-41 + GACGAAGAGACCCAGCAACCCA 22 2052 MLH1-42 + GAUGAUUGAGAACUGGUACGGA 22 2053 MLH1-43 − GCAAAGAGGCGUGUCCGCGCCA 22 2054 MLH1-44 − GCCAGUCAAAUUUCUCAACUCU 22 2055 MLH1-45 + GCGCCAAAAUGUCGUUCGUGGC 22 2056 MLH1-46 − GCGGCUACUGCCCGCUACCUAG 22 2057 MLH1-47 + GGGUUGUUUGGAGUGUAAGUGG 22 2058 MLH1-48 + GUCCAAUCAAUAGCUGCCGCUG 22 2059

Table II.70B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., MlH1 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the MlH1 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the MlH1 gene.

TABLE II.70B Exemplary HDR-enhancing gRNAs Targeting a MlH1 Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 70B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID MLH1-49 − AAUUUCUCAACUCUGUGGGUUG 22 2060 MLH1-50 + AUGCCCACAACGGCGGAGGCCG 22 2061 MLH1-51 + CCGAGACCUUUUAAGGGUUGUU 22 2062 MLH1-52 + CCGCUCUCCCCCGAGACCUUUU 22 2063 MLH1-53 − CGGCAGCUAUUGAUUGGACAGC 22 2064 MLH1-54 − CUUUGAUAGCAUUAGCUGGCCG 22 2065 MLH1-55 + UAGCUGCCGCUGAAGGGUGGGG 22 2066 MLH1-56 − UCACCACUGUCUCGUCCAGCCG 22 2067 MLH1-57 − UUGGUUCGUUUUUAGGAGCUCG 22 2068

Table II.71A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., PMS2 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the PMS2 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the PMS2 gene.

TABLE II.71A Exemplary HDR-enhancing gRNAs Targeting a PMS2 Gene S. pyogenes A high level of orthogonality, and starts with a G 1st Tier 71A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID PMS2-1 + GACAGAGCCAAUAGGCGAAA 20 2069 PMS2-2 − GACUGGGAAAGUUCCCUCCA 20 2070 PMS2-3 − GCAACACCCGAUCCGCCUCG 20 2071 PMS2-4 + GCAGCCAAUGGGAGUUCAGG 20 2072 PMS2-5 + GCCAAUGGGAGUUCAGGAGG 20 2073 PMS2-6 + GCCGCCCCGCCCGGAAAGGG 20 2074 PMS2-7 + GCGCCUGUGGGAGCCCUGGA 20 2075 PMS2-8 − GGACUGGGAAAGUUCCCUCC 20 2076 PMS2-9 + GGGAACUUUCCCAGUCCCCG 20 2077 PMS2-10 − GUGCUCCACCCUUUCCGGGC 20 2078 PMS2-11 − GUUCCCUCCAGGGCUCCCAC 20 2079

Table II.71B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., PMS2 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the PMS2 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the PMS2 gene.

TABLE II.71B Exemplary HDR-enhancing gRNAs Targeting a PMS2 Gene S. pyogenes A high level of orthogonality 2nd Tier 71B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID PMS2-12 + AAAGCAGCCAAUGGGAGUUC 20 2080 PMS2-13 − ACCCGAUCCGCCUCGGGGAC 20 2081 PMS2-14 + AGCGCCUGUGGGAGCCCUGG 20 2082 PMS2-15 + AGUAUUUUUGCCGCCCCGCC 20 2083 PMS2-16 − AUGCAACACCCGAUCCGCCU 20 2084 PMS2-17 − CCCGAUCCGCCUCGGGGACU 20 2085 PMS2-18 + CGACCUUUGACAGAGCCAAU 20 2086 PMS2-19 + CGGAGCGCCUGUGGGAGCCC 20 2087 PMS2-20 + CGGAUCGGGUGUUGCAUCCA 20 2088 PMS2-21 + CGGUGUGCUCUGAUUGGCCC 20 2089 PMS2-22 − CUUCGUGACGUCAAAGAGCC 20 2090 PMS2-23 + UCAGGAGGCGGAGCGCCUGU 20 2091 PMS2-24 + UCCCAGUCCCCGAGGCGGAU 20 2092 PMS2-25 − UCCGCCUCCUGAACUCCCAU 20 2093 PMS2-26 − UCGCCUAUUGGCUCUGUCAA 20 2094 PMS2-27 − UGCAACACCCGAUCCGCCUC 20 2095 PMS2-28 + UUCAGGAGGCGGAGCGCCUG 20 2096 PMS2-29 − UUCGUGACGUCAAAGAGCCU 20 2097 PMS2-30 + UUUGCCGCCCCGCCCGGAAA 20 2098 PMS2-31 + UUUUGCCGCCCCGCCCGGAA 20 2099

Table II.72A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., PMS2 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the PMS2 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the PMS2 gene.

TABLE II.72A Exemplary HDR-enhancing gRNA Targeting a PMS2 Gene S. aureus A high level of orthogonality,starts with a G, PAM is NNGRRT 1st Tier 72A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID PMS2-32 + GGGAACUUUCCCAGUCCCCGAG 22 2100

Table II.72B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., PMS2 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the PMS2 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the PMS2 gene.

TABLE II.72B Exemplary HDR-enhancing gRNAs Targeting a PMS2 Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 72B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID PMS2-33 + CACAACGUCGAAAGCAGCCAAU 22 2101 PMS2-34 + CUUUCCCAGUCCCCGAGGCGGA 22 2102 PMS2-35 + UAUUUUUGCCGCCCCGCCCGGA 22 2103

Table II.73A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., EZH2 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the EZH2 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the EZH2 gene.

TABLE II.73A Exemplary HDR-enhancing gRNAs Targeting a EZH2 Gene S. pyogenes A high level of orthogonality, and starts with a G 1st Tier 73A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID EZH2-1 + GAACAACGCGAGUCGGCGCG 20 2104 EZH2-2 + GACACCCGGUGGGACUCAGA 20 2105 EZH2-3 + GAGUGCGAACCGGGCGGCGG 20 2106 EZH2-4 + GCCACUGCUGUGCCGGUCCC 20 2107 EZH2-5 − GCCCCGAUUGGCGGGACGCG 20 2108 EZH2-6 + GCCGCGUUUGGCGCUCGGUC 20 2109 EZH2-7 + GCCUCGCGUCCCGCCAAUCG 20 2110 EZH2-8 + GCGGCGCUUGAUUGGGCUGG 20 2111 EZH2-9 − GCGGGCGCCCGUCCAAUCAC 20 2112 EZH2-10 − GGACCGGCACAGCAGUGGCG 20 2113 EZH2-11 + GGCGAUUGGGCUGCCGCGUU 20 2114 EZH2-12 + GGCGGCGCUUGAUUGGGCUG 20 2115 EZH2-13 + GGGCGGCGCUUGAUUGGGCU 20 2116 EZH2-14 + GGGCUCCGGGAGUGCGAACC 20 2117 EZH2-15 + GGGCUGCCGCGUUUGGCGCU 20 2118 EZH2-16 + GGGGCGGCGCUUGAUUGGGC 20 2119 EZH2-17 + GGGGCUCCGGGAGUGCGAAC 20 2120 EZH2-18 + GGGGGGCCAAAUAAAAGCGA 20 2121 EZH2-19 + GGUCGCGUCCGACACCCGGU 20 2122 EZH2-20 + GUCCGGUCGCGUCCGACACC 20 2123

Table II.73B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., EZH2 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the EZH2 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the EZH2 gene.

TABLE II.73B Exemplary HDR-enhancing gRNAs Targeting a EZH2 Gene S. pyogenes A high level of orthogonality 2nd Tier 73B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID EZH2-21 + AACAACGCGAGUCGGCGCGC 20 2124 EZH2-22 + AAUCGGGGCGGCGCUUGAUU 20 2125 EZH2-23 − ACCGGACCGAGCGCCAAACG 20 2126 EZH2-24 + ACGAAGGUAACGCGCCGCUG 20 2127 EZH2-25 + AGGUAACGCGCCGCUGCGGG 20 2128 EZH2-26 − CAAGCGCCGCCCCGAUUGGC 20 2129 EZH2-27 − CAAUCAAGCGCCGCCCCGAU 20 2130 EZH2-28 − CAAUCGCCAUCGCUUUUAUU 20 2131 EZH2-29 + CAAUCGGGGCGGCGCUUGAU 20 2132 EZH2-30 − CACCGGGUGUCGGACGCGAC 20 2133 EZH2-31 − CCGAUUGGCGGGACGCGAGG 20 2134 EZH2-32 + CCGCCUCGCGUCCCGCCAAU 20 2135 EZH2-33 + CGAGUCGGCGCGCGGGACGA 20 2136 EZH2-34 − CGAUUGGCGGGACGCGAGGC 20 2137 EZH2-35 − CGCCGCCCGGUUCGCACUCC 20 2138 EZH2-36 + CGCCUCGCGUCCCGCCAAUC 20 2139 EZH2-37 + CGCGCGGGAACAACGCGAGU 20 2140 EZH2-38 + CGGUCGCGUCCGACACCCGG 20 2141 EZH2-39 − UCAAGCGCCGCCCCGAUUGG 20 2142 EZH2-40 + UCGCGUCCCGCCAAUCGGGG 20 2143

Table II.74A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., EZH2 gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the EZH2 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the EZH2 gene.

TABLE II.74A Exemplary HDR-enhancing gRNAs Targeting a EZH2 Gene S. aureus A high level of orthogonality,starts with a G, PAM is NNGRRT 1st Tier 74A gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID EZH2-41 − GCCGCCGGGGCUCCACUGCCUU 22 2144 EZH2-42 + GCGGCCCGGCCGGCGGGGCUCC 22 2145 EZH2-43 + GGGGGCGACGCGCGGGAACAAC 22 2146

Table II.74B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., EZH2 gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the EZH2 gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the EZH2 gene.

TABLE II.74B Exemplary HDR-enhancing gRNAs Targeting a EZH2 Gene S. aureus A high level of orthogonality, and PAM is NNGRRT 2nd Tier 74B gRNA Target Site Name DNA Strand Targeting Domain Length Seq ID EZH2-44 + CCCCGCCACUGCUGUGCCGGUC 22 2147 EZH2-45 − CUCCACUGCCUUCUGAGUCCCA 22 2148

Table II.75A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., KDM4A (also referred to JMJD2A) gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the KDM4A (also referred to JMJD2A) gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the KDM4A (also referred to JMJD2A) gene.

TABLE II.75A Exemplary HDR-enhancing gRNAs Targeting a KDM4A Gene A high level of orthogonality, and starts with a G S. pyogenes 75A 1st Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID KDM4A-1 + GAGCUGAGCCUAAGCCCUGG 20 2149 KDM4A-2 + GAGUUUCGGCCUUCGCCUGC 20 2150 KDM4A-3 − GAUCCUACUGCUUUUCCAGC 20 2151 KDM4A-4 + GAUCGGCCAGUGGCGACAGC 20 2152 KDM4A-5 + GAUGCCGACUUUAGAGGAGG 20 2153 KDM4A-6 + GCAGAUGCCGACUUUAGAGG 20 2154 KDM4A-7 + GCUGAGCCUAAGCCCUGGCG 20 2155 KDM4A-8 + GCUUGCAGCCACCCUUGAAU 20 2156 KDM4A-9 + GGCUGUAGGUGAGAACUAUA 20 2157 KDM4A-10 + GGGCUGUAGGUGAGAACUAU 20 2158 KDM4A-11 − GUACAGAGUCAACCAAUUCA 20 2159

Table II.75B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., KDM4A (also referred to JMJD2A) gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the KDM4A (also referred to JMJD2A) gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the KDM4A (also referred to JMJD2A) gene.

TABLE II.75B Exemplary HDR-enhancing gRNAs Targeting a KDM4A Gene A high level of orthogonality S. pyogenes 75B 2nd Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID KDM4A-12 + AAAGCAGUAGGAUCGGCCAG 20 2160 KDM4A-13 − AACUCCGCCUCCUCUAAAGU 20 2161 KDM4A-14 − ACUGCUUUUCCAGCAGGCGA 20 2162 KDM4A-15 − AGAGUCAACCAAUUCAAGGG 20 2163 KDM4A-16 + AGCUGAGCCUAAGCCCUGGC 20 2164 KDM4A-17 − CCAAAGCCCCGCCAGGGCUU 20 2165 KDM4A-18 + CCUAAGCCCUGGCGGGGCUU 20 2166 KDM4A-19 + CGUGCUCAUUGGCUGGUGUA 20 2167 KDM4A-20 + CUAAGCCCUGGCGGGGCUUU 20 2168 KDM4A-21 − CUACAGCCCAAAGCCCCGCC 20 2169 KDM4A-22 + CUGGCGGGGCUUUGGGCUGU 20 2170 KDM4A-23 + CUUUAGAGGAGGCGGAGUUU 20 2171 KDM4A-24 − UACAGAGUCAACCAAUUCAA 20 2172 KDM4A-25 − UACAGCCCAAAGCCCCGCCA 20 2173 KDM4A-26 − UCAGCUCCUGCUGUCGCCAC 20 2174 KDM4A-27 + UCGCCUGCUGGAAAAGCAGU 20 2175 KDM4A-28 + UGCGCAGAUGCCGACUUUAG 20 2176 KDM4A-29 + UGCGGCGCGUGCUCAUUGGC 20 2177 KDM4A-30 + UGCUGGAAAAGCAGUAGGAU 20 2178 KDM4A-31 + UGGCUGCGGCGCGUGCUCAU 20 2179

Table II.76A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., KDM4A (also referred to JMJD2A) gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the KDM4A (also referred to JMJD2A) gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the KDM4A (also referred to JMJD2A) gene.

TABLE II.76A Exemplary HDR-enhancing gRNAs Targeting a KDM4A Gene A high level of orthogonality, starts with a G, PAM is NNGRRT S. aureus 76A 1st Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID KDM4A-32 − GCCGUACAGAGUCAACCAAUUC 22 2180 KDM4A-33 + GGUGUAUGGCUUGCAGCCACCC 22 2181

Table II.76B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., KDM4A (also referred to JMJD2A) gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the KDM4A (also referred to JMJD2A) gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the KDM4A (also referred to JMJD2A) gene.

TABLE II.76B Exemplary HDR-enhancing gRNAs Targeting a KDM4A Gene A high level of orthogonality, and PAM is NNGRRT S. aureus 76B 2nd Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID KDM4A-34 − AGUCGGCAUCUGCGCAGCCGUA 22 2182 KDM4A-35 + CAGAUGCCGACUUUAGAGGAGG 22 2183 KDM4A-36 + CCUUCGCCUGCUGGAAAAGCAG 22 2184 KDM4A-37 + UUUGGGCUGUAGGUGAGAACUA 22 2185

Table II.77A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., CDK gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and start with a 5′G. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the CDK gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the CDK gene.

TABLE II.77A Exemplary HDR-enhancing gRNAs Targeting a CDK Gene A high level of orthogonality, and starts with a G S. pyogenes 77A 1st Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID CDK1-1 + GAAGGCCUGCCCAGCGUAGC 20 2186 CDK1-2 + GAAUAAUAAGCCGGGUACAG 20 2187 CDK1-3 + GAAUCCGGGGCCCUUUAGCG 20 2188 CDK1-4 + GACGACACUCUCCCGACUGG 20 2189 CDK1-5 − GAGCGCGAAAGAAAGAGGAA 20 2190 CDK1-6 − GCAAGCGCUCUCCUCCAGUC 20 2191 CDK1-7 + GCCGCCGCGGAAUAAUAAGC 20 2192 CDK1-8 − GCGAAAGAAAGAGGAAAGGG 20 2193 CDK1-9 − GCGGCUAGAGAAAAAGCAGG 20 2194 CDK1-10 + GCUACCCGAUUGGUGAAUCC 20 2195 CDK1-11 + GCUGGCUCUUGGAAAUUGAG 20 2196 CDK1-12 − GCUGGGCAGGCCUUCCCGGG 20 2197 CDK1-13 + GGCUACCCGAUUGGUGAAUC 20 2198 CDK1-14 − GGCUAGAGCGCGAAAGAAAG 20 2199 CDK1-15 − GGGCCCCGGAUUCACCAAUC 20 2200 CDK1-16 + GGGGUCAGGGUCGUGUCUAG 20 2201 CDK1-17 + GGGUACAGUGGCUGGGGUCA 20 2202 CDK1-18 + GGUUGUUGUAGCUGCCGCUG 20 2203 CDK1-19 − GUACCCGGCUUAUUAUUCCG 20 2204 CDK1-20 − GUCCUACUGUUUCUAGUCAG 20 2205

Table II.77B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., CDK gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the CDK gene (e.g., a CDK1 gene). One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the CDK gene.

TABLE II.77B Exemplary HDR-enhancing gRNAs Targeting a CDK Gene A high level of orthogonality S. pyogenes 77B 2nd Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID CDK1-21 + AAAUUGAGCGGAGAGCGACG 20 2206 CDK1-22 + AAGUCUACGGGCUACCCGAU 20 2207 CDK1-23 + AGUUUGAAACUGCUCGCACU 20 2208 CDK1-24 − CAAUCAGAGCCCAGCUACGC 20 2209 CDK1-25 − CCCGGCUUAUUAUUCCGCGG 20 2210 CDK1-26 + CCGCCGCGGAAUAAUAAGCC 20 2211 CDK1-27 − CGCAAGCGCUCUCCUCCAGU 20 2212 CDK1-28 + CGCGCUCUAGCCACCCGGGA 20 2213 CDK1-29 + CGCUUGCGCUCGCACUCAGU 20 2214 CDK1-30 + CUACCCGAUUGGUGAAUCCG 20 2215 CDK1-31 − CUACGCUGGGCAGGCCUUCC 20 2216 CDK1-32 − CUCACCGCGCUAAAGGGCCC 20 2217 CDK1-33 + CUCCGCUGACUAGAAACAGU 20 2218 CDK1-34 + CUUUCGCGCUCUAGCCACCC 20 2219 CDK1-35 + UAGGACGACACUCUCCCGAC 20 2220 CDK1-36 + UCUUUCGCGCUCUAGCCACC 20 2221 CDK1-37 + UGGGGUCAGGGUCGUGUCUA 20 2222 CDK1-38 − UUAUUCCGCGGCGGCCGCAG 20 2223 CDK1-39 − UUCAAACUCACCGCGCUAAA 20 2224 CDK1-40 − UUUCAAACUCACCGCGCUAA 20 2225

Table II.78A provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., CDK gene. The targeting domains of gRNAs were selected according to the first tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality, start with a 5′G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the CDK gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the CDK gene.

TABLE II.78A Exemplary HDR-enhancing gRNAs Targeting a CDK Gene A high level of orthogonality, starts with a G, PAM is NNGRRT S. aureus 78A 1st Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID CDK1-41 + GAAUCCGGGGCCCUUUAGCGCG 22 2226 CDK1-42 − GCAAGCGCUCUCCUCCAGUCGG 22 2227 CDK1-43 + GCGGCCGCCGCGGAAUAAUAAG 22 2228 CDK1-44 + GUAGCUGCCGCUGCGGCCGCCG 22 2229

Table II.78B provides exemplary targeting domains of gRNAs to be used with an eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) a gene, e.g., CDK gene. The targeting domains of gRNAs were selected according to the second tier parameters. The targeting domains bind within 500 bp spanning a transcription start site (TSS), e.g., upstream or downstream of a TSS, have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 fusion molecule (e.g., an eiCas9 fused to a transcription activator or repressor domain) to alter (e.g., activate or repress) the CDK gene. One or more gRNA may be used to target an eiCas9 fusion molecule to a region spanning 500 bp of a transcription start site (TSS) of the CDK gene.

TABLE II.78B Exemplary HDR-enhancing gRNAs Targeting a CDK Gene A high level of orthogonality, and PAM is NNGRRT S. aureus 78B 2nd Tier Target Site gRNA Name DNA Strand Targeting Domain Length Seq ID CDK1-45 − AAACUCACCGCGCUAAAGGGCC 22 2230 CDK1-46 − AAAGCAGGAGGGCGGGCGCCAA 22 2231 CDK1-47 + AAGUCUACGGGCUACCCGAUUG 22 2232 CDK1-48 + AGCCGGGUACAGUGGCUGGGGU 22 2233 CDK1-49 + AUAAUAAGCCGGGUACAGUGGC 22 2234 CDK1-50 − CAGCUACGCUGGGCAGGCCUUC 22 2235 CDK1-51 − UAAAGGGCCCCGGAUUCACCAA 22 2236

III. Cas9 Molecules

Cas9 molecules of a variety of species can be used in the methods and compositions described herein. While the S. pyogenes, S. aureus, and S. thermophilus Cas9 molecules are the subject of much of the disclosure herein, Cas9 molecules, derived from, or based on the Cas9 proteins of other species listed herein can be used as well. In other words, while the much of the description herein uses S. pyogenes and S. thermophilus Cas9 molecules, Cas9 molecules from the other species can replace them, e.g., Staphylococcus aureus and Neisseria meningitidis Cas9 molecules. Additional Cas9 species include: Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhizobium sp., Brevibacillus laterosporus, Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Candidatus Puniceispirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, gamma proteobacterium, Gluconacetobacter diazotrophicus, Haemophilus parainfluenzae, Haemophilus sputorum, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, Ilyobacter polytropus, Kingella kingae, Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp., Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans, Pasteurella multocida, Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus lugdunensis, Streptococcus sp., Subdoligranulum sp., Tistrella mobilis, Treponema sp., or Verminephrobacter eiseniae.

A Cas9 molecule, or Cas9 polypeptide, as the term is used herein, refers to a molecule or a polypeptide that can interact with a guide RNA (gRNA) molecule) and, in concert with the gRNA molecule, localizes to a site which comprises a target domain, and in some embodiments, a PAM sequence. Cas9 molecule and Cas9 polypeptide, as those terms are used herein, refer to naturally occurring Cas9 molecules and to engineered, altered, or modified Cas9 molecules or Cas9 polypeptides that differ, e.g., by at least one amino acid residue, from a reference sequence, e.g., the most similar naturally occurring Cas9 molecule or a sequence of Table III.1.

Cas9 Domains

Crystal structures have been determined for two different naturally occurring bacterial Cas9 molecules (Jinek et al., SCIENCE, 343(6176): 1247997, 2014) and for S. pyogenes Cas9 with a guide RNA (e.g., a synthetic fusion of crRNA and tracrRNA) (Nishimasu et al., CELL, 156:935-949, 2014; and Anders et al., NATURE, 2014, doi: 10.1038/nature13579).

A naturally occurring Cas9 molecule comprises two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe; each of which further comprise domains described herein. The REC lobe comprises the arginine-rich bridge helix (BH), the REC1 domain, and the REC2 domain. The REC lobe does not share structural similarity with other known proteins, indicating that it is a Cas9-specific functional domain. The BH domain is a long a helix and arginine rich region and comprises amino acids 60-93 of the sequence of S. pyogenes Cas9. The REC1 domain is important for recognition of the repeat:anti-repeat duplex, e.g., of a gRNA or a tracrRNA, and is therefore critical for Cas9 activity by recognizing the target sequence. The REC1 domain comprises two REC1 motifs at amino acids 94 to 179 and 308 to 717 of the sequence of S. pyogenes Cas9. These two REC1 domains, though separated by the REC2 domain in the linear primary structure, assemble in the tertiary structure to form the REC1 domain. The REC2 domain, or parts thereof, may also play a role in the recognition of the repeat:anti-repeat duplex. The REC2 domain comprises amino acids 180-307 of the sequence of S. pyogenes Cas9.

The NUC lobe comprises the RuvC domain, the HNH domain, and the PAM-interacting (PI) domain. The RuvC domain shares structural similarity to retroviral integrase superfamily members and cleaves a single strand, e.g., the non-complementary strand of the target nucleic acid molecule. The RuvC domain is assembled from the three split RuvC motifs (RuvC I, RuvCII, and RuvCIII, which are often commonly referred to in the art as RuvCI domain, or N-terminal RuvC domain, RuvCII domain, and RuvCIII domain) at amino acids 1-59, 718-769, and 909-1098, respectively, of the sequence of S. pyogenes Cas9. Similar to the REC1 domain, the three RuvC motifs are linearly separated by other domains in the primary structure, however in the tertiary structure, the three RuvC motifs assemble and form the RuvC domain. The HNH domain shares structural similarity with HNH endonucleases, and cleaves a single strand, e.g., the complementary strand of the target nucleic acid molecule. The HNH domain lies between the RuvC II-III motifs and comprises amino acids 775-908 of the sequence of S. pyogenes Cas9. The PI domain interacts with the PAM of the target nucleic acid molecule, and comprises amino acids 1099-1368 of the sequence of S. pyogenes Cas9.

RuvC-Like Domain and HNH-Like Domain

In an embodiment, a Cas9 molecule or Cas9 polypeptide comprises an HNH-like domain and a RuvC-like domain. In an embodiment, cleavage activity is dependent on a RuvC-like domain and an HNH-like domain. A Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, can comprise one or more of the following domains: a RuvC-like domain and an HNH-like domain. In an embodiment, a Cas9 molecule or Cas9 polypeptide is an eaCas9 molecule or eaCas9 polypeptide and the eaCas9 molecule or eaCas9 polypeptide comprises a RuvC-like domain, e.g., a RuvC-like domain described below, and/or an HNH-like domain, e.g., an HNH-like domain described below.

RuvC-Like Domains

In an embodiment, a RuvC-like domain cleaves, a single strand, e.g., the non-complementary strand of the target nucleic acid molecule. The Cas9 molecule or Cas9 polypeptide can include more than one RuvC-like domain (e.g., one, two, three or more RuvC-like domains). In an embodiment, a RuvC-like domain is at least 5, 6, 7, 8 amino acids in length but not more than 20, 19, 18, 17, 16 or 15 amino acids in length. In an embodiment, the Cas9 molecule or Cas9 polypeptide comprises an N-terminal RuvC-like domain of about 10 to 20 amino acids, e.g., about 15 amino acids in length.

N-Terminal RuvC-Like Domains

Some naturally occurring Cas9 molecules comprise more than one RuvC-like domain with cleavage being dependent on the N-terminal RuvC-like domain. Accordingly, Cas9 molecules or Cas9 polypeptide can comprise an N-terminal RuvC-like domain. Exemplary N-terminal RuvC-like domains are described below.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an N-terminal RuvC-like domain comprising an amino acid sequence of formula I:

-   -   D-X1-G-X2-X3-X4-X5-G-X6-X7-X8-X9, wherein,     -   X1 is selected from I, V, M, L and T (e.g., selected from I, V,         and L);     -   X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected         from T, V, and I);     -   X3 is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);     -   X4 is selected from S, Y, N and F (e.g., S);     -   X5 is selected from V, I, L, C, T and F (e.g., selected from V,         I and L);     -   X6 is selected from W, F, V, Y, S and L (e.g., W);     -   X7 is selected from A, S, C, V and G (e.g., selected from A and         S);     -   X8 is selected from V, I, L, A, M and H (e.g., selected from V,         I, M and L); and     -   X9 is selected from any amino acid or is absent (e.g., selected         from T, V, I, L, A, F, S, A, Y, M and R, or, e.g., selected from         T, V, I, L and A).

In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID NO: 108, by as many as 1 but no more than 2, 3, 4, or 5 residues.

In embodiment, the N-terminal RuvC-like domain is cleavage competent.

In embodiment, the N-terminal RuvC-like domain is cleavage incompetent.

In an embodiment, a eaCas9 molecule or eaCas9 polypeptide comprises an N-terminal RuvC-like domain comprising an amino acid sequence of formula II:

(SEQ ID NO: 109), D-X1-G-X2-X3-S-X5-G-X6-X7-X8-X9,

wherein

-   -   X1 is selected from I, V, M, L and T (e.g., selected from I, V,         and L);     -   X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected         from T, V, and I);     -   X3 is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);     -   X5 is selected from V, I, L, C, T and F (e.g., selected from V,         I and L);     -   X6 is selected from W, F, V, Y, S and L (e.g., W);     -   X7 is selected from A, S, C, V and G (e.g., selected from A and         S);     -   X8 is selected from V, I, L, A, M and H (e.g., selected from V,         I, M and L); and     -   X9 is selected from any amino acid or is absent (e.g., selected         from T, V, I, L, A, F, S, A, Y, M and R or selected from e.g.,         T, V, I, L and A).

In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID NO: 109 by as many as 1 but no more than 2, 3, 4, or 5 residues.

In an embodiment, the N-terminal RuvC-like domain comprises an amino acid sequence of formula III:

(SEQ ID NO: 110) D-I-G-X2-X3-S-V-G-W-A-X8-X9,

wherein

-   -   X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected         from T, V, and I);     -   X3 is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);     -   X8 is selected from V, I, L, A, M and H (e.g., selected from V,         I, M and L); and     -   X9 is selected from any amino acid or is absent (e.g., selected         from T, V, I, L, A, F, S, A, Y, M and R or selected from e.g.,         T, V, I, L and A).

In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID NO:110 by as many as 1 but no more than, 2, 3, 4, or 5 residues.

In an embodiment, the N-terminal RuvC-like domain comprises an amino acid sequence of formula III:

(SEQ ID NO: 111) D-I-G-T-N-S-V-G-W-A-V-X,

wherein

-   -   X is a non-polar alkyl amino acid or a hydroxyl amino acid,         e.g., X is selected from V, I, L and T.

In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID NO: 111 by as many as 1 but no more than, 2, 3, 4, or 5 residues.

In an embodiment, the N-terminal RuvC-like domain differs from a sequence of an N-terminal RuvC like domain disclosed herein, as many as 1 but no more than 2, 3, 4, or 5 residues.

Additional RuvC-Like Domains

In addition to the N-terminal RuvC-like domain, the Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, can comprise one or more additional RuvC-like domains. In an embodiment, the Cas9 molecule or Cas9 polypeptide can comprise two additional RuvC-like domains. Preferably, the additional RuvC-like domain is at least 5 amino acids in length and, e.g., less than 15 amino acids in length, e.g., 5 to 10 amino acids in length, e.g., 8 amino acids in length.

An additional RuvC-like domain can comprise an amino acid sequence:

(SEQ ID NO: 112) I-X1-X2-E-X3-A-R-E, wherein

-   -   X1 is V or H,     -   X2 is I, L or V (e.g., I or V); and     -   X3 is M or T.

In an embodiment, the additional RuvC-like domain comprises the amino acid sequence:

(SEQ ID NO: 113) I-V-X2-E-M-A-R-E, wherein

-   -   X2 is I, L or V (e.g., I or V).

An additional RuvC-like domain can comprise an amino acid sequence:

(SEQ ID NO: 114) H-H-A-X1-D-A-X2-X3, wherein

-   -   X1 is H or L;     -   X2 is R or V; and     -   X3 is E or V.

In an embodiment, the additional RuvC-like domain comprises the amino acid sequence:

(SEQ ID NO: 115) H-H-A-H-D-A-Y-L.

In an embodiment, the additional RuvC-like domain differs from a sequence of SEQ ID NOS 112, 113, 114, and 115 by as many as 1 but no more than 2, 3, 4, or 5 residues.

In some embodiments, the sequence flanking the N-terminal RuvC-like domain is a sequences of formula V:

(SEQ ID NO: 116 K-X1′-Y-X2′-X3′-X4′-Z-T-D-X9′-Y,  and 2290, respectively, in order of appearance).

wherein

-   -   X1′ is selected from K and P,     -   X2′ is selected from V, L, I, and F (e.g., V, I and L);     -   X3′ is selected from G, A and S (e.g., G),     -   X4′ is selected from L, I, V and F (e.g., L);     -   X9′ is selected from D, E, N and Q; and     -   Z is an N-terminal RuvC-like domain, e.g., as described above.

HNH-Like Domains

In an embodiment, an HNH-like domain cleaves a single stranded complementary domain, e.g., a complementary strand of a double stranded nucleic acid molecule. In an embodiment, an HNH-like domain is at least 15, 20, 25 amino acids in length but not more than 40, 35 or 30 amino acids in length, e.g., 20 to 35 amino acids in length, e.g., 25 to 30 amino acids in length. Exemplary HNH-like domains are described below.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an HNH-like domain having an amino acid sequence of formula VI:

(SEQ ID NO: 117) X1-X2-X3-H-X4-X5-P-X6-X7-X8-X9-X10-X11-X12-X13- X14-X15-N-X16-X17-X18-X19-X20-X21-X22-X23-N, wherein

-   -   X1 is selected from D, E, Q and N (e.g., D and E);     -   X2 is selected from L, I, R, Q, V, M and K;     -   X3 is selected from D and E;     -   X4 is selected from I, V, T, A and L (e.g., A, I and V);     -   X5 is selected from V, Y, I, L, F and W (e.g., V, I and L);     -   X6 is selected from Q, H, R, K, Y, I, L, F and W;     -   X7 is selected from S, A, D, T and K (e.g., S and A);     -   X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g.,         F);     -   X9 is selected from L, R, T, I, V, S, C, Y, K, F and G;     -   X10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;     -   X11 is selected from D, S, N, R, L and T (e.g., D);     -   X12 is selected from D, N and S;     -   X13 is selected from S, A, T, G and R (e.g., S);     -   X14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g.,         I, L and F);     -   X15 is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and         V;     -   X16 is selected from K, L, R, M, T and F (e.g., L, R and K);     -   X17 is selected from V, L, I, A and T;     -   X18 is selected from L, I, V and A (e.g., L and I);     -   X19 is selected from T, V, C, E, S and A (e.g., T and V);     -   X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H         and A;     -   X21 is selected from S, P, R, K, N, A, H, Q, G and L;     -   X22 is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y;         and     -   X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D         and F.

In an embodiment, a HNH-like domain differs from a sequence of SEQ ID NO: 117 by at least one but no more than, 2, 3, 4, or 5 residues.

In an embodiment, the HNH-like domain is cleavage competent.

In an embodiment, the HNH-like domain is cleavage incompetent.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an HNH-like domain comprising an amino acid sequence of formula VII:

(SEQ ID NO: 118) X1-X2-X3-H-X4-X5-P-X6-S-X8-X9-X10-D-D-S-X14-X15- N-K-V-L-X19-X20-X21-X22-X23-N,

wherein

-   -   X1 is selected from D and E;     -   X2 is selected from L, I, R, Q, V, M and K;     -   X3 is selected from D and E;     -   X4 is selected from I, V, T, A and L (e.g., A, I and V);     -   X5 is selected from V, Y, I, L, F and W (e.g., V, I and L);     -   X6 is selected from Q, H, R, K, Y, I, L, F and W;     -   X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g.,         F);     -   X9 is selected from L, R, T, I, V, S, C, Y, K, F and G;     -   X10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;     -   X14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g.,         I, L and F);     -   X15 is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and         V;     -   X19 is selected from T, V, C, E, S and A (e.g., T and V);     -   X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H         and A;     -   X21 is selected from S, P, R, K, N, A, H, Q, G and L;     -   X22 is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y;         and     -   X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D         and F.

In an embodiment, the HNH-like domain differs from a sequence of SEQ ID NO: 118 by 1, 2, 3, 4, or 5 residues.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an HNH-like domain comprising an amino acid sequence of formula VII:

(SEQ ID NO: 119) X1-V-X3-H-I-V-P-X6-S-X8-X9-X10-D-D-S-X14-X15-N-K- V-L-T-X20-X21-X22-X23-N, wherein

-   -   X1 is selected from D and E;     -   X3 is selected from D and E;     -   X6 is selected from Q, H, R, K, Y, I, L and W;     -   X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g.,         F);     -   X9 is selected from L, R, T, I, V, S, C, Y, K, F and G;     -   X10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;     -   X14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g.,         I, L and F);     -   X15 is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and         V;     -   X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H         and A;     -   X21 is selected from S, P, R, K, N, A, H, Q, G and L;     -   X22 is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y;         and     -   X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D         and F.

In an embodiment, the HNH-like domain differs from a sequence of SEQ ID NO: 119 by 1, 2, 3, 4, or 5 residues.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an HNH-like domain having an amino acid sequence of formula VIII:

(SEQ ID NO: 120) D-X2-D-H-I-X5-P-Q-X7-F-X9-X10-D-X12-S-I-D-N-X16-V- L-X19-X20-S-X22-X23-N,

wherein

-   -   X2 is selected from I and V;     -   X5 is selected from I and V;     -   X7 is selected from A and S;     -   X9 is selected from I and L;     -   X10 is selected from K and T;     -   X12 is selected from D and N;     -   X16 is selected from R, K and L; X19 is selected from T and V;     -   X20 is selected from S and R;     -   X22 is selected from K, D and A; and     -   X23 is selected from E, K, G and N (e.g., the eaCas9 molecule or         eaCas9 polypeptide can comprise an HNH-like domain as described         herein).

In an embodiment, the HNH-like domain differs from a sequence of SEQ ID NO: 120 by as many as 1 but no more than 2, 3, 4, or 5 residues.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises the amino acid sequence of formula IX:

(SEQ ID NO: 121 L-Y-Y-L-Q-N-G-X1′-D-M-Y-X2′-X3′-X4′-X5′-L-D-I-X6′- X7′-L-S-X8′-Y-Z-N-R-X9′-K-X10′-D-X11′-V-P and 2291, respectively, in order of appearance),

wherein

-   -   X1′ is selected from K and R;     -   X2′ is selected from V and T;     -   X3′ is selected from G and D;     -   X4′ is selected from E, Q and D;     -   X5′ is selected from E and D;     -   X6′ is selected from D, N and H;     -   X7′ is selected from Y, R and N;     -   X8′ is selected from Q, D and N; X9′ is selected from G and E;     -   X10′ is selected from S and G;     -   X11′ is selected from D and N; and     -   Z is an HNH-like domain, e.g., as described above.

In an embodiment, the eaCas9 molecule or eaCas9 polypeptide comprises an amino acid sequence that differs from a sequence of SEQ ID NO: 121 by as many as 1 but no more than 2, 3, 4, or 5 residues.

In an embodiment, the HNH-like domain differs from a sequence of an HNH-like domain disclosed herein, by as many as 1 but no more than 2, 3, 4, or 5 residues.

In an embodiment, the HNH-like domain differs from a sequence of an HNH-like domain disclosed herein, by as many as 1 but no more than 2, 3, 4, or 5 residues.

Cas9 Activities

Nuclease and Helicase Activities

In an embodiment, the Cas9 molecule or Cas9 polypeptide is capable of cleaving a target nucleic acid molecule. Typically wild type Cas9 molecules cleave both strands of a target nucleic acid molecule. Cas9 molecules and Cas9 polypeptides can be engineered to alter nuclease cleavage (or other properties), e.g., to provide a Cas9 molecule or Cas9 polypeptide which is a nickase, or which lacks the ability to cleave target nucleic acid. A Cas9 molecule or Cas9 polypeptide that is capable of cleaving a target nucleic acid molecule is referred to herein as an eaCas9 (an enzymatically active Cas9) molecule or eaCas9 polypeptide.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises one or more of the following enzymatic activities:

-   -   a nickase activity, i.e., the ability to cleave a single strand,         e.g., the non-complementary strand or the complementary strand,         of a nucleic acid molecule;     -   a double stranded nuclease activity, i.e., the ability to cleave         both strands of a double stranded nucleic acid and create a         double strand break, which in an embodiment is the presence of         two nickase activities;     -   an endonuclease activity;     -   an exonuclease activity; and     -   a helicase activity, i.e., the ability to unwind the helical         structure of a double stranded nucleic acid.

In an embodiment, an enzymatically active or an eaCas9 molecule or eaCas9 polypeptide cleaves both DNA strands and results in a double strand break. In an embodiment, an eaCas9 molecule or eaCas9 polypeptide cleaves only one strand, e.g., the strand to which the gRNA hybridizes to, or the strand complementary to the strand the gRNA hybridizes with. In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises cleavage activity associated with an HNH domain. In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises cleavage activity associated with a RuvC domain. In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises cleavage activity associated with an HNH domain and cleavage activity associated with a RuvC domain. In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an active, or cleavage competent, HNH domain and an inactive, or cleavage incompetent, RuvC domain. In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an inactive, or cleavage incompetent, HNH domain and an active, or cleavage competent, RuvC domain.

Some Cas9 molecules or Cas9 polypeptides have the ability to interact with a gRNA molecule, and in conjunction with the gRNA molecule, localize to a target sequence on a target nucleic acid (the target domain), but are incapable of cleaving the target nucleic acid, or incapable of cleaving at efficient rates. Cas9 molecules having no, or no substantial, cleavage activity are referred to herein as an eiCas9 molecule or eiCas9 polypeptide. For example, an eiCas9 molecule or eiCas9 polypeptide can lack cleavage activity or have substantially less, e.g., less than 20, 10, 5, 1 or 0.1% of the cleavage activity of a reference Cas9 molecule or eiCas9 polypeptide, as measured by an assay described herein.

Targeting and PAMs

A Cas9 molecule or Cas9 polypeptide, is a polypeptide that can interact with a guide RNA (gRNA) molecule and, in concert with the gRNA molecule, localizes to a site which comprises a target domain, and in an embodiment, a PAM sequence.

In an embodiment, the ability of an eaCas9 molecule or eaCas9 polypeptide to interact with and cleave a target nucleic acid is PAM sequence dependent. A PAM sequence is a sequence in the target nucleic acid. In an embodiment, cleavage of the target nucleic acid occurs upstream from the PAM sequence. EaCas9 molecules from different bacterial species can recognize different sequence motifs (e.g., PAM sequences). In an embodiment, an eaCas9 molecule of S. pyogenes recognizes the sequence motif NGG and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. See, e.g., Mali et al., SCIENCE (2013) 339(6121): 823-826. In an embodiment, an eaCas9 molecule of S. thermophilus recognizes the sequence motif NGGNG and/or NNAGAAW (W=A or T) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from these sequences. See, e.g., Horvath et al., SCIENCE (2010); 327(5962):167-170, and Deveau et al., J. BACTERIOL. 2008; 190(4): 1390-1400. In an embodiment, an eaCas9 molecule of S. mutans recognizes the sequence motif NGG and/or NAAR (R=A or G) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5 base pairs, upstream from this sequence. See, e.g., Deveau et al., J BACTERIOL 2008; 190(4): 1390-1400. In an embodiment, an eaCas9 molecule of S. aureus recognizes the sequence motif NNGRR (R=A or G) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. In an embodiment, an eaCas9 molecule of S. aureus recognizes the sequence motif NNGRRN (R=A or G) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. In an embodiment, an eaCas9 molecule of S. aureus recognizes the sequence motif NNGRRT (R=A or G) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. In an embodiment, an eaCas9 molecule of S. aureus recognizes the sequence motif NNGRRV (R=A or G) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. In an embodiment, an eaCas9 molecule of N. meningitidis recognizes the sequence motif NNNNGATT or NNNGCTT (R=A or G) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. See, e.g., Hou et al. (2013) PROC. NAT′L. ACAD. SCI. USA 110(39):15644-15649. The ability of a Cas9 molecule to recognize a PAM sequence can be determined, e.g., using a transformation assay described in Jinek et al. (2012) SCIENCE 337:816. In the aforementioned embodiments, N can be any nucleotide residue, e.g., any of A, G, C or T. In one embodiment, the PAM sequence is facing outward.

As is discussed herein, Cas9 molecules can be engineered to alter the PAM specificity of the Cas9 molecule.

Exemplary naturally occurring Cas9 molecules are described in Chylinski et al. (2013) RNA BIOLOGY 10:5, 727-737. Such Cas9 molecules include Cas9 molecules of a cluster 1 bacterial family, cluster 2 bacterial family, cluster 3 bacterial family, cluster 4 bacterial family, cluster 5 bacterial family, cluster 6 bacterial family, a cluster 7 bacterial family, a cluster 8 bacterial family, a cluster 9 bacterial family, a cluster 10 bacterial family, a cluster 11 bacterial family, a cluster 12 bacterial family, a cluster 13 bacterial family, a cluster 14 bacterial family, a cluster 15 bacterial family, a cluster 16 bacterial family, a cluster 17 bacterial family, a cluster 18 bacterial family, a cluster 19 bacterial family, a cluster 20 bacterial family, a cluster 21 bacterial family, a cluster 22 bacterial family, a cluster 23 bacterial family, a cluster 24 bacterial family, a cluster 25 bacterial family, a cluster 26 bacterial family, a cluster 27 bacterial family, a cluster 28 bacterial family, a cluster 29 bacterial family, a cluster 30 bacterial family, a cluster 31 bacterial family, a cluster 32 bacterial family, a cluster 33 bacterial family, a cluster 34 bacterial family, a cluster 35 bacterial family, a cluster 36 bacterial family, a cluster 37 bacterial family, a cluster 38 bacterial family, a cluster 39 bacterial family, a cluster 40 bacterial family, a cluster 41 bacterial family, a cluster 42 bacterial family, a cluster 43 bacterial family, a cluster 44 bacterial family, a cluster 45 bacterial family, a cluster 46 bacterial family, a cluster 47 bacterial family, a cluster 48 bacterial family, a cluster 49 bacterial family, a cluster 50 bacterial family, a cluster 51 bacterial family, a cluster 52 bacterial family, a cluster 53 bacterial family, a cluster 54 bacterial family, a cluster 55 bacterial family, a cluster 56 bacterial family, a cluster 57 bacterial family, a cluster 58 bacterial family, a cluster 59 bacterial family, a cluster 60 bacterial family, a cluster 61 bacterial family, a cluster 62 bacterial family, a cluster 63 bacterial family, a cluster 64 bacterial family, a cluster 65 bacterial family, a cluster 66 bacterial family, a cluster 67 bacterial family, a cluster 68 bacterial family, a cluster 69 bacterial family, a cluster 70 bacterial family, a cluster 71 bacterial family, a cluster 72 bacterial family, a cluster 73 bacterial family, a cluster 74 bacterial family, a cluster 75 bacterial family, a cluster 76 bacterial family, a cluster 77 bacterial family, or a cluster 78 bacterial family.

Exemplary naturally occurring Cas9 molecules include a Cas9 molecule of a cluster 1 bacterial family. Examples include a Cas9 molecule of: S. pyogenes (e.g., strain SF370, MGAS10270, MGAS10750, MGAS2096, MGAS315, MGAS5005, MGAS6180, MGAS9429, NZ131 and SSI-1), S. thermophilus (e.g., strain LMD-9), S. pseudoporcinus (e.g., strain SPIN 20026), S. mutans (e.g., strain UA159, NN2025), S. macacae (e.g., strain NCTC11558), S. gallolyticus (e.g., strain UCN34, ATCC BAA-2069), S. equines (e.g., strain ATCC 9812, MGCS 124), S. dysdalactiae (e.g., strain GGS 124), S. bovis (e.g., strain ATCC 700338), S. anginosus (e.g., strain F0211), S. agalactiae (e.g., strain NEM316, A909), Listeria monocytogenes (e.g., strain F6854), Listeria innocua (L. innocua, e.g., strain Clip11262), Enterococcus italicus (e.g., strain DSM 15952), or Enterococcus faecium (e.g., strain 1,231,408). Additional exemplary Cas9 molecules are a Cas9 molecule of Neisseria meningitidis (Hou et al., PNAS Early Edition 2013, 1-6 and a S. aureus Cas9 molecule. In an embodiment, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, comprises an amino acid sequence:

-   -   having 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,         87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or         99% homology with;     -   differs at no more than, 2, 5, 10, 15, 20, 30, or 40% of the         amino acid residues when compared with;     -   differs by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,         14, 15, 16, 17, 18, 19, 20 amino acids, but by no more than 100,         80, 70, 60, 50, 40 or 30 amino acids from; or     -   is identical to any Cas9 molecule sequence described herein, or         a naturally occurring Cas9 molecule sequence, e.g., a Cas9         molecule from a species listed herein or described in Chylinski         et al. (2013) RNA BIOLOGY 10:5, 727-737; Hou et al., PNAS Early         Edition 2013, 1-6. In an embodiment, the Cas9 molecule or Cas9         polypeptide comprises one or more of the following activities: a         nickase activity; a double stranded cleavage activity (e.g., an         endonuclease and/or exonuclease activity); a helicase activity;         or the ability, together with a gRNA molecule, to localize to a         target nucleic acid.

Engineered or Altered Cas9 Molecules and Cas9 Polypeptides

Cas9 molecules and Cas9 polypeptides described herein, e.g., naturally occurring Cas9 molecules, can possess any of a number of properties, including: nuclease activity (e.g., endonuclease and/or exonuclease activity); helicase activity; the ability to associate functionally with a gRNA molecule; and the ability to target (or localize to) a site on a nucleic acid (e.g., PAM recognition and specificity). In an embodiment, a Cas9 molecule or Cas9 polypeptide can include all or a subset of these properties. In a typical embodiment, a Cas9 molecule or Cas9 polypeptide has the ability to interact with a gRNA molecule and, in concert with the gRNA molecule, localize to a site in a nucleic acid. Other activities, e.g., PAM specificity, cleavage activity, or helicase activity can vary more widely in Cas9 molecules and Cas9 polypeptides.

Cas9 molecules include engineered Cas9 molecules and engineered Cas9 polypeptides (engineered, as used in this context, means merely that the Cas9 molecule or Cas9 polypeptide differs from a reference sequences, and implies no process or origin limitation). An engineered Cas9 molecule or Cas9 polypeptide can comprise altered enzymatic properties, e.g., altered nuclease activity (as compared with a naturally occurring or other reference Cas9 molecule) or altered helicase activity. As discussed herein, an engineered Cas9 molecule or Cas9 polypeptide can have nickase activity (as opposed to double strand nuclease activity). In an embodiment an engineered Cas9 molecule or Cas9 polypeptide can have an alteration that alters its size, e.g., a deletion of amino acid sequence that reduces its size, e.g., without significant effect on one or more, or any Cas9 activity. In an embodiment, an engineered Cas9 molecule or Cas9 polypeptide can comprise an alteration that affects PAM recognition. For example, an engineered Cas9 molecule can be altered to recognize a PAM sequence other than that recognized by the endogenous wild-type PI domain. In an embodiment a Cas9 molecule or Cas9 polypeptide can differ in sequence from a naturally occurring Cas9 molecule but not have significant alteration in one or more Cas9 activities.

Cas9 molecules or Cas9 polypeptides with desired properties can be made in a number of ways, e.g., by alteration of a parental, e.g., naturally occurring Cas9 molecule or Cas9 polypeptide, to provide an altered Cas9 molecule or Cas9 polypeptide having a desired property. For example, one or more mutations or differences relative to a parental Cas9 molecule, e.g., a naturally occurring or engineered Cas9 molecule, can be introduced. Such mutations and differences comprise: substitutions (e.g., conservative substitutions or substitutions of non-essential amino acids), insertions, or deletions. In an embodiment, a Cas9 molecule or Cas9 polypeptide can comprises one or more mutations or differences, e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or 50 mutations but less than 200, 100, or 80 mutations relative to a reference, e.g., a parental Cas9 molecule.

In an embodiment, a mutation or mutations do not have a substantial effect on a Cas9 activity, e.g. a Cas9 activity described herein. In an embodiment, a mutation or mutations have a substantial effect on a Cas9 activity, e.g. a Cas9 activity described herein.

Non-Cleaving and Modified-Cleavage Cas9 Molecules and Cas9 Polypeptides

In an embodiment, a Cas9 molecule or Cas9 polypeptide comprises a cleavage property that differs from naturally occurring Cas9 molecules, e.g., that differs from the naturally occurring Cas9 molecule having the closest homology. For example, a Cas9 molecule or Cas9 polypeptide can differ from a naturally occurring Cas9 molecule, e.g., a Cas9 molecule of S. pyogenes, as follows: its ability to modulate, e.g., decreased or increased, cleavage of a double stranded nucleic acid (endonuclease and/or exonuclease activity), e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S. pyogenes); its ability to modulate, e.g., decreased or increased, cleavage of a single strand of a nucleic acid, e.g., a non-complementary strand of a nucleic acid molecule or a complementary strand of a nucleic acid molecule (nickase activity), e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S. pyogenes); or the ability to cleave a nucleic acid molecule, e.g., a double stranded or single stranded nucleic acid molecule, can be eliminated.

Alterations in the Ability to Cleave One or Both Strands of a Target Nucleic Acid

In an embodiment, exemplary Cas9 activities comprise one or more of PAM specificity, cleavage activity, and helicase activity. A mutation(s) can be present, e.g., in: one or more RuvC domains, e.g., an N-terminal RuvC domain; an HNH domain; a region outside the RuvC domains and the HNH domain. In an embodiment, a mutation(s) is present in a RuvC domain. In an embodiment, a mutation(s) is present in an HNH domain. In an embodiment, mutations are present in both a RuvC domain and an HNH domain.

Exemplary mutations that may be made in the RuvC domain or HNH domain with reference to the S. pyogenes Cas9 sequence include: D10A, E762A, H840A, N854A, N863A and/or D986A. Exemplary mutations that may be made in the RuvC domain with reference to the S. aureus Cas9 sequence include: N580A.

In an embodiment, a Cas9 molecule is an eiCas9 molecule comprising one or more differences in a RuvC domain and/or in an HNH domain as compared to a reference Cas9 molecule, and the eiCas9 molecule does not cleave a nucleic acid, or cleaves with significantly less efficiency than does wild type, e.g., when compared with wild type in a cleavage assay, e.g., as described herein, cuts with less than 50, 25, 10, or 1% of a reference Cas9 molecule, as measured by an assay described herein.

Whether or not a particular sequence, e.g., a substitution, may affect one or more activity, such as targeting activity, cleavage activity, etc., can be evaluated or predicted, e.g., by evaluating whether the mutation is conservative. In an embodiment, a “non-essential” amino acid residue, as used in the context of a Cas9 molecule, is a residue that can be altered from the wild-type sequence of a Cas9 molecule, e.g., a naturally occurring Cas9 molecule, e.g., an eaCas9 molecule, without abolishing or more preferably, without substantially altering a Cas9 activity (e.g., cleavage activity), whereas changing an “essential” amino acid residue results in a substantial loss of activity (e.g., cleavage activity).

In an embodiment, a Cas9 molecule comprises a cleavage property that differs from naturally occurring Cas9 molecules, e.g., that differs from the naturally occurring Cas9 molecule having the closest homology. For example, a Cas9 molecule can differ from naturally occurring Cas9 molecules, e.g., a Cas9 molecule of S. aureus, S. pyogenes, or C. jejuni as follows: its ability to modulate, e.g., decreased or increased, cleavage of a double strand break (endonuclease and/or exonuclease activity), e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S. aureus, S. pyogenes, or C. jejuni); its ability to modulate, e.g., decreased or increased, cleavage of a single strand of a nucleic acid, e.g., a non-complimentary strand of a nucleic acid molecule or a complementary strand of a nucleic acid molecule (nickase activity), e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S. aureus, S. pyogenes, or C. jejuni); or the ability to cleave a nucleic acid molecule, e.g., a double stranded or single stranded nucleic acid molecule, can be eliminated.

In an embodiment, the altered Cas9 molecule is an eaCas9 molecule comprising one or more of the following activities: cleavage activity associated with a RuvC domain; cleavage activity associated with an HNH domain; cleavage activity associated with an HNH domain and cleavage activity associated with a RuvC domain.

In an embodiment, the altered Cas9 molecule is an eiCas9 molecule which does not cleave a nucleic acid molecule (either double stranded or single stranded nucleic acid molecules) or cleaves a nucleic acid molecule with significantly less efficiency, e.g., less than 20, 10, 5, 1 or 0.1% of the cleavage activity of a reference Cas9 molecule, e.g., as measured by an assay described herein. The reference Cas9 molecule can be a naturally occurring unmodified Cas9 molecule, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S. pyogenes, S. thermophilus, S. aureus, C. jejuni or N. meningitidis. In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology. In an embodiment, the eiCas9 molecule lacks substantial cleavage activity associated with a RuvC domain and cleavage activity associated with an HNH domain.

In an embodiment, the altered Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, can be a fusion, e.g., of two of more different Cas9 molecules, e.g., of two or more naturally occurring Cas9 molecules of different species. For example, a fragment of a naturally occurring Cas9 molecule of one species can be fused to a fragment of a Cas9 molecule of a second species. As an example, a fragment of a Cas9 molecule of S. pyogenes comprising an N-terminal RuvC-like domain can be fused to a fragment of Cas9 molecule of a species other than S. pyogenes (e.g., S. thermophilus) comprising an HNH-like domain.

Cas9 Molecules with Altered PAM Recognition or No PAM Recognition

Naturally occurring Cas9 molecules can recognize specific PAM sequences, for example the PAM recognition sequences described above for, e.g., S. pyogenes, S. thermophilus, S. mutans, S. aureus and N. meningitidis.

In an embodiment, a Cas9 molecule or Cas9 polypeptide has the same PAM specificities as a naturally occurring Cas9 molecule. In an embodiment, a Cas9 molecule or Cas9 polypeptide has a PAM specificity not associated with a naturally occurring Cas9 molecule, or a PAM specificity not associated with the naturally occurring Cas9 molecule to which it has the closest sequence homology. For example, a naturally occurring Cas9 molecule can be altered, e.g., to alter PAM recognition, e.g., to alter the PAM sequence that the Cas9 molecule or Cas9 polypeptide recognizes to decrease off target sites and/or improve specificity; or eliminate a PAM recognition requirement. In an embodiment, a Cas9 molecule or Cas9 polypeptide can be altered, e.g., to increase length of PAM recognition sequence and/or improve Cas9 specificity to a high level of identity (e.g., 98%, 99% or 100% match between gRNA and a PAM sequence), e.g., to decrease off target sites and increase specificity. In an embodiment, the length of the PAM recognition sequence is at least 4, 5, 6, 7, 8, 9, 10 or 15 amino acids in length. Cas9 molecules or Cas9 polypeptides that recognize different PAM sequences and/or have reduced off-target activity can be generated using directed evolution. Exemplary methods and systems that can be used for directed evolution of Cas9 molecules are described, e.g., in Esvelt et al. (2011) NATURE 472(7344): 499-503. Candidate Cas9 molecules can be evaluated, e.g., by methods described in Section IV.

In one embodiment, the Cas9 molecule is a S. pyogenes Cas9 variant. In certain embodiments, the Cas9 variant is the EQR variant. In certain embodiments, the Cas9 variant is the VRER variant. In certain embodiments, the eiCas9 molecule is a S. pyogenes Cas9 variant. In certain embodiments, the Cas9 variant is the EQR variant. In certain embodiments, the Cas9 variant is the VRER variant. Cas9 variants are described, for example, in Kleinstiver et al., Nature, 523:481-485, 2015.

In certain embodiments, a Cas9 system comprises a Cas9 molecule, e.g., a Cas9 molecule described herein, e.g., the Cas9 EQR variant or the Cas9 VRER variant.

Following identification, gRNAs can be ranked into tiers based on their distance to the target site, their orthogonality and presence of a 5′ G (based on identification of close matches in the human genome containing a relevant PAM (e.g., for a S. pyogenes Cas9 EQR variant, the PAM may be a NGAG PAM, A NGCG PAM, a NGGG PAM, a NGTG PAM, a NGAA PAM, a NGAT PAM or a NGAC PAM).

Following identification, gRNAs can be ranked into tiers based on their distance to the target site, their orthogonality and presence of a 5′ G (based on identification of close matches in the human genome containing a relevant PAM (e.g., for a S. pyogenes Cas9 VRER variant, the PAM may be a NGCG PAM, A NGCA PAM, a NGCT PAM, or a NGCC PAM).

In some embodiments, the Cas9 molecule is a S. aureus Cas9 variant. In certain embodiments, the Cas9 variant is the KKH (E782K/N968K/R1015H) variant (see Kleinstiver et al. (2015) NAT. BIOTECHNOL. doi: 10.1038/nbt.3404, the entire contents of which are expressly incorporated herein by reference). In some embodiments, the Cas9 variant is the E782K/K929R/R1015H variant (see Kleinstiver et al. (2015)). In some embodiments, the Cas9 variant is the E782K/K929R/N968K/R1015H variant (see Kleinstiver et al. (2015). In some embodiments the Cas9 variant comprises one or more mutations in one of the following residues: E782, K929, N968, R1015. In some embodiments the Cas9 variant comprises one or more of the following mutations: E782K, K929R, N968K, R1015H and R1015Q (see Kleinstiver et al. (2015)). In certain embodiments, a Cas9 system comprises a Cas9 molecule, e.g., a Cas9 molecule described herein, e.g., the Cas9 KKH variant.

Following identification, gRNAs can be ranked into tiers based on their distance to the target site, their orthogonality and presence of a 5′ G (based on identification of close matches in the human genome containing a relevant PAM (e.g., for a S. aureus Cas9 KKH variant, the PAM may be a NNNRRT PAM (e.g., a NNNAGT PAM, a NNNGGT PAM, a NNNGAT PAM, or a NNNAAT PAM).

Alterations of the PI domain, which mediates PAM recognition are discussed below.

Synthetic Cas9 Molecules and Cas9 Polypeptides with Altered PI Domains

Current genome-editing methods are limited in the diversity of target sequences that can be targeted by the PAM sequence that is recognized by the Cas9 molecule utilized. A synthetic Cas9 molecule (or Syn-Cas9 molecule), or synthetic Cas9 polypeptide (or syn-Cas9 polypeptide), as that term is used herein, refers to a Cas9 molecule or Cas9 polypeptide that comprises a Cas9 core domain from one bacterial species and a functional altered PI domain, i.e., a PI domain other than that naturally associated with the Cas9 core domain, e.g., from a different bacterial species.

In an embodiment, the altered PI domain recognizes a PAM sequence that is different from the PAM sequence recognized by the naturally-occurring Cas9 from which the Cas9 core domain is derived. In an embodiment, the altered PI domain recognizes the same PAM sequence recognized by the naturally-occurring Cas9 from which the Cas9 core domain is derived, but with different affinity or specificity. A Syn-Cas9 molecule or Syn-Cas9 polypeptide can be, respectively, a Syn-eaCas9 molecule or Syn-eaCas9 polypeptide or a Syn-eiCas9 molecule Syn-eiCas9 polypeptide.

An exemplary Syn-Cas9 molecule Syn-Cas9 polypeptide comprises:

-   -   a) a Cas9 core domain, e.g., a Cas9 core domain from Table III.1         or 3, e.g., a S. aureus, S. pyogenes, or C. jejuni Cas9 core         domain; and     -   b) an altered PI domain from a species X Cas9 sequence selected         from Tables 111.4 and III.5.

In an embodiment, the RKR motif (the PAM binding motif) of said altered PI domain comprises: differences at 1, 2, or 3 amino acid residues; a difference in amino acid sequence at the first, second, or third position; differences in amino acid sequence at the first and second positions, the first and third positions, or the second and third positions; as compared with the sequence of the RKR motif of the native or endogenous PI domain associated with the Cas9 core domain.

In an embodiment, the Cas9 core domain comprises the Cas9 core domain from a species X Cas9 from Table III.1 and said altered PI domain comprises a PI domain from a species Y Cas9 from Table III.1.

In an embodiment, the RKR motif of the species X Cas9 is other than the RKR motif of the species Y Cas9.

In an embodiment, the RKR motif of the altered PI domain is selected from XXY, XNG, and XNQ.

In an embodiment, the altered PI domain has at least 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homology with the amino acid sequence of a naturally occurring PI domain of said species Y from Table III.1.

In an embodiment, the altered PI domain differs by no more than 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue from the amino acid sequence of a naturally occurring PI domain of said second species from Table III.1.

In an embodiment, the Cas9 core domain comprises a S. aureus core domain and altered PI domain comprises: an A. denitrificans PI domain; a C. jejuni PI domain; a H. mustelae PI domain; or an altered PI domain of species X PI domain, wherein species X is selected from Table III.4 or Table III.5.

In an embodiment, the Cas9 core domain comprises a S. pyogenes core domain and the altered PI domain comprises: an A. denitrificans PI domain; a C. jejuni PI domain; a H. mustelae PI domain; or an altered PI domain of species X PI domain, wherein species X is selected from Table III.4 or Table II.5.

In an embodiment, the Cas9 core domain comprises a C. jejuni core domain and the altered PI domain comprises: an A. denitrificans PI domain; a H. mustelae PI domain; or an altered PI domain of species X PI domain, wherein species X is selected from Table III.4 or Table III.5.

In an embodiment, the Cas9 molecule further comprises a linker disposed between said Cas9 core domain and said altered PI domain.

In an embodiment, the linker comprises: a linker described elsewhere herein disposed between the Cas9 core domain and the heterologous PI domain.

Exemplary altered PI domains for use in Syn-Cas9 molecules are described in Tables III.4 and III.5. The sequences for the 83 Cas9 orthologs referenced in Tables 111.4 and 111.5 are provided in Table III.1. Table III.2 provides the Cas9 orthologs with known PAM sequences and the corresponding RKR motif.

In an embodiment, a Syn-Cas9 molecule may also be size-optimized, e.g., the Syn-Cas9 molecule comprises one or more deletions, and optionally one or more linkers disposed between the amino acid residues flanking the deletions. In an embodiment, a Syn-Cas9 molecule comprises a REC deletion.

Size-Optimized Cas9 Molecules

Engineered Cas9 molecules and engineered Cas9 polypeptides, as described herein, include a Cas9 molecule or Cas9 polypeptide comprising a deletion that reduces the size of the molecule while still retaining desired Cas9 properties, e.g., essentially native conformation, Cas9 nuclease activity, and/or target nucleic acid molecule recognition. Provided herein are Cas9 molecules or Cas9 polypeptides comprising one or more deletions, and optionally one or more linkers, wherein a linker is disposed between the amino acid residues that flank the deletion. Methods for identifying suitable deletions in a reference Cas9 molecule, methods for generating Cas9 molecules with a deletion and a linker, and methods for using such Cas9 molecules will be apparent to one of ordinary skill in the art upon review of this document.

A Cas9 molecule, e.g., a S. aureus, S. pyogenes, or C. jejuni, Cas9 molecule, having a deletion is smaller, e.g., has reduced number of amino acids, than the corresponding naturally-occurring Cas9 molecule. The smaller size of the Cas9 molecules allows increased flexibility for delivery methods, and thereby increases utility for genome-editing. A Cas9 molecule can comprise one or more deletions that do not substantially affect or decrease the activity of the resultant Cas9 molecules described herein. Activities that are retained in the Cas9 molecules comprising a deletion as described herein include one or more of the following:

-   -   a nickase activity, i.e., the ability to cleave a single strand,         e.g., the non-complementary strand or the complementary strand,         of a nucleic acid molecule; a double stranded nuclease activity,         i.e., the ability to cleave both strands of a double stranded         nucleic acid and create a double strand break, which in an         embodiment is the presence of two nickase activities;     -   an endonuclease activity;     -   an exonuclease activity;     -   a helicase activity, i.e., the ability to unwind the helical         structure of a double stranded nucleic acid;     -   and recognition activity of a nucleic acid molecule, e.g., a         target nucleic acid or a gRNA.

Activity of the Cas9 molecules described herein can be assessed using the activity assays described herein or in the art.

Identifying Regions Suitable for Deletion

Suitable regions of Cas9 molecules for deletion can be identified by a variety of methods. Naturally-occurring orthologous Cas9 molecules from various bacterial species, e.g., any one of those listed in Table III.1, can be modeled onto the crystal structure of S. pyogenes Cas9 (Nishimasu et al. (2014) CELL, 156: 935-949) to examine the level of conservation across the selected Cas9 orthologs with respect to the three-dimensional conformation of the protein. Less conserved or unconserved regions that are located spatially distant from regions involved in Cas9 activity, e.g., the interface with a target nucleic acid molecule and/or gRNA, represent regions or domains that are candidates for deletion without substantially affecting or decreasing Cas9 activity.

REC-Optimized Cas9 Molecules

A REC-optimized Cas9 molecule, as that term is used herein, refers to a Cas9 molecule that comprises a deletion in one or both of the REC2 domain and the RE1_(CT) domain (collectively a REC deletion), wherein the deletion comprises at least 10% of the amino acid residues in the cognate domain. A REC-optimized Cas9 molecule can be an eaCas9 molecule or an eiCas9 molecule. An exemplary REC-optimized Cas9 molecule comprises:

-   -   a) a deletion selected from:         -   i) a REC2 deletion;         -   ii) a REC1_(CT) deletion; or         -   iii) a REC1_(SUB) deletion.

Optionally, a linker is disposed between the amino acid residues that flank the deletion. In an embodiment a Cas9 molecule includes only one deletion, or only two deletions. A Cas9 molecule can comprise a REC2 deletion and a REC1_(CT) deletion. A Cas9 molecule can comprise a REC2 deletion and a REC1_(SUB) deletion.

Generally, the deletion will contain at least 10% of the amino acids in the cognate domain, e.g., a REC2 deletion will include at least 10% of the amino acids in the REC2 domain.

A deletion can comprise: at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% of the amino acid residues of its cognate domain; all of the amino acid residues of its cognate domain; an amino acid residue outside its cognate domain; a plurality of amino acid residues outside its cognate domain; the amino acid residue immediately N terminal to its cognate domain; the amino acid residue immediately C terminal to its cognate domain; the amino acid residue immediately N terminal to its cognate and the amino acid residue immediately C terminal to its cognate domain; a plurality of, e.g., up to 5, 10, 15, or 20, amino acid residues N terminal to its cognate domain; a plurality of, e.g., up to 5, 10, 15, or 20, amino acid residues C terminal to its cognate domain; a plurality of, e.g., up to 5, 10, 15, or 20, amino acid residues N terminal to its cognate domain and a plurality of e.g., up to 5, 10, 15, or 20, amino acid residues C terminal to its cognate domain.

In an embodiment, a deletion does not extend beyond: its cognate domain; the N terminal amino acid residue of its cognate domain; the C terminal amino acid residue of its cognate domain.

A REC-optimized Cas9 molecule can include a linker disposed between the amino acid residues that flank the deletion. Linkers for use in generating recombinant proteins, e.g., multi-domain proteins, are known in the art (Chen et al. (2013) ADV. DRUG DELIVERYREV. 65:1357-69). Any linkers known in the art that maintain the conformation or native fold of the Cas9 molecule (thereby retaining Cas9 activity) can be used. Several properties of linkers, such as length, hydrophobicity, intrinsic properties of the amino acids residues themselves, and secondary structure should be considered in the context of the goal to maintain native conformation and functional activity of Cas9. Any linkers known in the art that maintain the conformation or native fold of the Cas9 molecule (thereby retaining Cas9 activity) can be used. Several properties of linkers, such as length, hydrophobicity, intrinsic properties of the amino acids residues themselves, and secondary structure should be considered in the context of the goal to maintain native conformation and functional activity of Cas9.

A flexible linker can be utilized in the Cas9 molecules described herein. Flexible linkers allow a certain degree of movement and/or interaction within and between the joined domains or regions of the protein. Generally, flexible linkers are composed of small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The small size of these amino acids provides flexibility and allows mobility of the connected domains or regions. Furthermore, the incorporation of Ser or Thr can help maintain the stability of the linker in aqueous solutions by hydrogen bonding with the water molecules, thereby reducing unfavorable interactions between the linker and the other protein moieties. Commonly used flexible linkers are comprised of sequences that primarily consist of Gly and Ser residues. Often, these flexible linkers consist of repeating units of a combination of Gly and Ser residues, e.g., (GGS)_(x), where the number of repeating units, e.g., x, can be optimized to achieve the appropriate separation of other domains or regions of the protein.

In some cases, a rigid linker may be preferred if there is significant distance between the joined domains or regions, or to maintain a fixed distance between the joined domains or regions of a protein and independent functions of the domains/regions. Rigid linkers often have defined secondary structure, e.g., alpha helix, or other stabilizing interactions, e.g., salt bridges and disulfide bonds. Rigid linkers commonly contain multiple Pro residues, or repeating combinations of Glu-Pro or Lys-Pro because Pro imposes a strong conformation constraint due to its structure.

The linker can comprise an amino acid residue, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. Typically, the linker will comprises less than 10, 20 or 30 amino acid residues. Typically, the linker is less than 50, 40, 30, 20, 10, or 5% of the length of the deleted sequence. Suitable linkers include: [Gly-Ser]_(x), wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 341); [Gly-Gly-Ser]_(x), wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 339); [Gly-Gly-Ser]; [Gly-Ser-Gly-Ser]_(x), wherein x is 1, 2, 3, 4, or 5 (SEQ ID NO: 127); [Gly-Ser-Gly-Ser] (SEQ ID NO: 128); (GSAGSAAGSGEF)_(x), wherein x is 1, 2, 3 or 4 (SEQ ID NO: 201); (SIVAQLSRPDPA) x(SEQ ID NO: 202), wherein x is 1, 2, 3 or 4; or an XTEN sequence, e.g., the XTEN sequence of SEQ ID NO: #______, or a sequence that differs therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues. In an embodiment linker comprises an amino acid sequence other than a sequence within REC2.

In an embodiment, a REC-optimized Cas9 molecule comprises an amino acid sequence that, other than any REC deletion and associated linker, has at least 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology with the amino acid sequence of a naturally occurring Cas9, e.g., a Cas9 molecule described in Table III.1, e.g., a S. aureus Cas9 molecule, a S. pyogenes Cas9 molecule, or a C. jejuni Cas9 molecule.

In an embodiment, a REC-optimized Cas9 molecule comprises an amino acid sequence that, other than any REC deletion and associated linker, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid residues from the amino acid sequence of a naturally occurring Cas9, e.g., a Cas9 molecule described in Table III.1, e.g., a S. aureus Cas9 molecule, a S. pyogenes Cas9 molecule, or a C. jejuni Cas9 molecule.

In an embodiment, a REC-optimized Cas9 molecule comprises an amino acid sequence that, other than any REC deletion and associate linker, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25% of the amino acid residues from the amino acid sequence of a naturally occurring Cas9, e.g., a Cas9 molecule described in Table III.1, e.g., a S. aureus Cas9 molecule, a S. pyogenes Cas9 molecule, or a C. jejuni Cas9 molecule.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) ADV. APPL. MATH. 2: 482c, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. MOL. BIOL. 48:443, by the search for similarity method of Pearson and Lipman, (1988) PROC. NAT′L. ACAD. SCI. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Brent et al., (2003) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY).

Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) NUC. ACIDS RES. 25: 3389-3402; and Altschul et al. (1990) J. MOL. BIOL. 215: 403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (1988) COMPUT. APPL. BIOSCI. 4:11-17, which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (1970) J. MOL. BIOL. 48:444-453 algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

Sequence information for exemplary REC deletions are provided for 83 naturally-occurring Cas9 orthologs in Table III.1.

The amino acid sequences of exemplary Cas9 molecules from different bacterial species are shown below.

TABLE III.1 Amino Acid Sequence of Cas9 Orthologs REC2 REC1_(CT) REC_(sub) start stop # AA start stop # AA start stop # AA Amino acid (AA (AA deleted (AA (AA deleted (AA (AA deleted Species/Composite ID sequence pos) pos) (n) pos) pos) (n) pos) pos) (n) Staphylococcus aureus SEQ ID NO: 6 126 166 41 296 352 57 296 352 57 tr|J7RUA5|J7RUA5_STAAU Streptococcus pyogenes SEQ ID NO: 7 176 314 139 511 592 82 511 592 82 sp|Q99ZW2|CAS9_STRP1 Campylobacter jejuni NCTC SEQ ID NO: 8 137 181 45 316 360 45 316 360 45 11168 gi|218563121|ref|YP_002344900.1 Bacteroides fragilis NCTC 9343 SEQ ID NO: 9 148 339 192 524 617 84 524 617 84 gi|60683389|ref|YP_213533.1| Bifidobacterium bifidum S17 SEQ ID NO: 173 335 163 516 607 87 516 607 87 gi|310286728|ref|YP_003937986. 10 Veillonella atypica ACS-134-V- SEQ ID NO: 185 339 155 574 663 79 574 663 79 Col7a 11 gi|303229466|ref|ZP_07316256.1 Lactobacillus rhamnosus GG SEQ ID NO: 169 320 152 559 645 78 559 645 78 gi|258509199|ref|YP_003171950.1 12 Filifactor alocis ATCC 35896 SEQ ID NO: 166 314 149 508 592 76 508 592 76 gi|374307738|ref|YP_005054169.1 13 Oenococcus kitaharae DSM SEQ ID NO: 169 317 149 555 639 80 555 639 80 17330 14 gi|366983953|gb|EHN59352.1| Fructobacillus fructosus KCTC SEQ ID NO: 168 314 147 488 571 76 488 571 76 3544 15 gi|339625081|ref|ZP_08660870.1 Catenibacterium mitsuokai DSM SEQ ID NO: 173 318 146 511 594 78 511 594 78 15897 16 gi|224543312|ref|ZP_03683851.1 Finegoldia magna ATCC 29328 SEQ ID NO: 168 313 146 452 534 77 452 534 77 gi|169823755|ref|YP_001691366.1 17 CoriobacteriumglomeransPW2 SEQ ID NO: 175 318 144 511 592 82 511 592 82 gi|328956315|ref|YP_004373648.1 18 Eubacterium yurii ATCC 43715 SEQ ID NO: 169 310 142 552 633 76 552 633 76 gi|306821691|ref|ZP_07455288.1 19 Peptoniphilus duerdenii ATCC SEQ ID NO: 171 311 141 535 615 76 535 615 76 BAA-1640 20 gi|1304438954|ref|ZP_07398877.1 Acidaminococcus sp. D21 SEQ ID NO: 167 306 140 511 591 75 511 591 75 gi|1227824983|ref|ZP_03989815.1 21 Lactobacillus farciminis KCTC SEQ ID NO: 171 310 140 542 621 85 542 621 85 3681 22 gi|336394882|ref|ZP_08576281.1 Streptococcus sanguinis SK49 SEQ ID NO: 185 324 140 411 490 85 411 490 85 gi|422884106|ref|ZP_16930555.1 23 Coprococcus catus GD-7 SEQ ID NO: 172 310 139 556 634 76 556 634 76 gi|291520705|emb|CBK78998.1| 24 Streptococcus mutans UA159 SEQ ID NO: 176 314 139 392 470 84 392 470 84 gi|24379809|ref|NP_721764.1| 25 Streptococcus pyogenes M1 GAS SEQ ID NO: 176 314 139 523 600 82 523 600 82 gi|13622193|gb|AAK33936.1| 26 Streptococcus thermophilus SEQ ID NO: 176 314 139 481 558 81 481 558 81 LMD-9 27 gi|116628213|ref|YP_820832.1| Fusobacteriumnucleatum SEQ ID NO: 171 308 138 537 614 76 537 614 76 ATCC49256 28 gi|34762592|ref|ZP_00143587.1| Planococcus antarcticus DSM SEQ ID NO: 162 299 138 538 614 94 538 614 94 14505 29 gi|389815359|ref|ZP_10206685.1 Treponema denticola ATCC SEQ ID NO: 169 305 137 524 600 81 524 600 81 35405 30 gi|42525843|ref|NP_970941.1| Solobacterium moorei F0204 SEQ ID NO: 179 314 136 544 619 77 544 619 77 gi|320528778|ref|ZP_08029929.1 31 Staphylococcus SEQ ID NO: 164 299 136 531 606 92 531 606 92 pseudintermedius ED99 32 gi|323463801|gb|ADX75954.1| Flavobacterium branchiophilum SEQ ID NO: 162 286 125 538 613 63 538 613 63 FL-15 33 gi|1347536497|ref|YP_004843922.1 Ignavibacterium album JCM SEQ ID NO: 223 329 107 357 432 90 357 432 90 16511 34 gi|385811609|ref|YP_005848005.1 Bergeyella zoohelcum ATCC SEQ ID NO: 165 261 97 529 604 56 529 604 56 43767 35 gi|423317190|ref|ZP_17295095.1 Nitrobacter hamburgensis X14 SEQ ID NO: 169 253 85 536 611 48 536 611 48 gi|92109262|ref|YP_571550.1| 36 Odoribacter laneus YIT 12061 SEQ ID NO: 164 242 79 535 610 63 535 610 63 gi|374384763|ref|ZP_09642280.1 37 Legionella pneumophila str. Paris SEQ ID NO: 164 239 76 402 476 67 402 476 67 gi|54296138|ref|YP_122507.1| 38 Bacteroides sp. 20 3 SEQ ID NO: 198 269 72 530 604 83 530 604 83 gi|301311869|ref|ZP_07217791.1 39 Akkermansia muciniphila ATCC SEQ ID NO: 136 202 67 348 418 62 348 418 62 BAA-835 40 gi|187736489|ref|YP_001878601. Prevotella sp. C561 SEQ ID NO: 184 250 67 357 425 78 357 425 78 gi|345885718|ref|ZP_08837074.1 41 Wolinella succinogenes DSM SEQ ID NO: 157 218 36 401 468 60 401 468 60 1740 42 gi|34557932|ref|NP_907747.1| Alicyclobacillus hesperidum SEQ ID NO: 142 196 55 416 482 61 416 482 61 URH17-3-68 43 gi|403744858|ref|ZP_10953934.1 Caenispirillum salinarum AK4 SEQ ID NO: 161 214 54 330 393 68 330 393 68 gi|427429481|ref|ZP_18919511.1 44 Eubacterium rectale ATCC 33656 SEQ ID NO: 133 185 53 322 384 60 322 384 60 gi|238924075|ref|YP_002937591.1 45 Mycoplasma synoviae 53 SEQ ID NO: 187 239 53 319 381 80 319 381 80 gi|71894592|ref|YP_278700.1| 46 Porphyromonas sp. oral taxon SEQ ID NO: 150 202 53 309 371 60 309 371 60 279 str. F0450 47 gi|402847315|ref|ZP_10895610.1 Streptococcus thermophilus SEQ ID NO: 127 178 139 424 486 81 424 486 81 LMD-9 48 gi|116627542|ref|YP_820161.1| Roseburia inulinivorans DSM SEQ ID NO: 154 204 51 318 380 69 318 380 69 16841 49 gi|225377804|ref|ZP_03755025.1 Methylosinus trichosporium SEQ ID NO: 144 193 50 426 488 64 426 488 64 OB3b 50 gi|296446027|ref|ZP_06887976.1 Ruminococcus albus 8 SEQ ID NO: 139 187 49 351 412 55 351 412 55 gi|325677756|ref|ZP_08157403.1 51 Bifidobacterium longum DJO10A SEQ ID NO: 183 230 48 370 431 44 370 431 44 gi|189440764|ref|YP_001955845. 52 Enterococcus faecalis TX0012 SEQ ID NO: 123 170 48 327 387 60 327 387 60 gi|315149830|gb|EFT93846.1| 53 Mycoplasma mobile 163K SEQ ID NO: 179 226 48 314 374 79 314 374 79 gi|47458868|ref|YP_015730.1| 54 Actinomyces coleocanis DSM SEQ ID NO: 147 193 47 358 418 40 358 418 40 15436 55 gi|227494853|ref|ZP_03925169.1 Dinoroseobacter shibae DFL 12 SEQ ID NO: 138 184 47 338 398 48 338 398 48 gi|159042956|ref|YP_001531750.1 56 Actinomyces sp. oral taxon 180 SEQ ID NO: 183 228 46 349 409 40 349 409 40 str. F0310 57 gi|315605738|ref|ZP_07880770.1 Alcanivorax sp. W11-5 SEQ ID NO: 139 183 45 344 404 61 344 404 61 gi|407803669|ref|ZP_11150502.1 58 Aminomonas paucivorans DSM SEQ ID NO: 134 178 45 341 401 63 341 401 63 12260 59 gi|312879015|ref|ZP_07738815.1 Mycoplasma canis PG 14 SEQ ID NO: 139 183 45 319 379 76 319 379 76 gi|384393286|gb|EIE39736.1| 60 Lactobacillus coryniformis KCTC SEQ ID NO: 141 184 44 328 387 61 328 387 61 3535 61 gi|336393381|ref|ZP_08574780.1 Elusimicrobium minutum Pei191 SEQ ID NO: 177 219 43 322 381 47 322 381 47 gi|187250660|ref|YP_001875142.1 62 Neisseria meningitidis Z2491 SEQ ID NO: 147 189 43 360 419 61 360 419 61 gi|218767588|ref|YP_002342100.1 63 Pasteurella multocida str. Pm70 SEQ ID NO: 139 181 43 319 378 61 319 378 61 gi|15602992|ref|NP_246064.1| 64 Rhodovulum sp. PH10 SEQ ID NO: 141 183 43 319 378 48 319 378 48 gi|402849997|ref|ZP_10898214.1 65 Eubacterium dolichum DSM SEQ ID NO: 131 172 42 303 361 59 303 361 59 3991 66 gi|160915782|ref|ZP_02077990.1 Nitratifractor salsuginis DSM SEQ ID NO: 143 184 42 347 404 61 347 404 61 16511 67 gi|319957206|ref|YP_004168469.1 Rhodospirillum rubrum ATCC SEQ ID NO: 139 180 42 314 371 55 314 371 55 11170 68 gi|83591793|ref|YP_425545.1| Clostridium cellulolyticum H10 SEQ ID NO: 137 176 40 320 376 61 320 376 61 gi|220930482|ref|YP_002507391.1 69 Helicobacter mustelae 12198 SEQ ID NO: 148 187 40 298 354 48 298 354 48 gi|291276265|ref|YP_003516037.1 70 Ilyobacter polytropus DSM 2926 SEQ ID NO: 134 173 40 462 517 63 462 517 63 gi|310780384|ref|YP_003968716.1 71 Sphaerochaeta globus str. Buddy SEQ ID NO: 163 202 40 335 389 45 335 389 45 gi|325972003|ref|YP_004248194.1 72 Staphylococcus lugdunensis SEQ ID NO: 128 167 40 337 391 57 337 391 57 M23590 73 gi|315659848|ref|ZP_07912707.1 Treponema sp. JC4 SEQ ID NO: 144 183 40 328 382 63 328 382 63 gi|384109266|ref|ZP_10010146.1 74 uncultured delta SEQ ID NO: 154 193 40 313 365 55 313 365 55 proteobacterium HF0070 07E19 75 gi|297182908|gb|ADI19058.1| Alicycliphilus denitrificans K601 SEQ ID NO: 140 178 39 317 366 48 317 366 48 gi|330822845|ref|YP_004386148.1 76 Azospirillum sp. B510 SEQ ID NO: 205 243 39 342 389 46 342 389 46 gi|288957741|ref|YP_003448082.1 77 Bradyrhizobium sp. BTAi1 SEQ ID NO: 143 181 39 323 370 48 323 370 48 gi|148255343|ref|YP_001239928.1 78 Parvibaculum lavamentivorans SEQ ID NO: 138 176 39 327 374 58 327 374 58 DS-1 79 gi|154250555|ref|YP_001411379.1 Prevotella timonensis CRIS 5C-B1 SEQ ID NO: 170 208 39 328 375 61 328 375 61 gi|282880052|ref|ZP_06288774.1 80 Bacillus smithii 7 3 47FAA SEQ ID NO: 134 171 38 401 448 63 401 448 63 gi|365156657|ref|ZP_09352959.1 81 Cand. Puniceispirillum marinum SEQ ID NO: 135 172 38 344 391 53 344 391 53 IMCC1322 82 gi|294086111|ref|YP_003552871.1 Barnesiella intestinihominis YIT SEQ ID NO: 140 176 37 371 417 60 371 417 60 11860 83 gi|404487228|ref|ZP_11022414.1 Ralstonia syzygii R24 SEQ ID NO: 140 176 37 395 440 50 395 440 50 gi|344171927|emb|CCA84553.1| 84 Wolinella succinogenes DSM SEQ ID NO: 145 180 36 348 392 60 348 392 60 1740 86 gi|34557790|ref|NP_907605.1| Mycoplasma gallisepticum str. F SEQ ID NO: 144 177 34 373 416 71 373 416 71 gi|284931710|gb|ADC31648.1| 87 Acidothermus cellulolyticus 11B SEQ ID NO: 150 182 33 341 380 58 341 380 58 gi|117929158|ref|YP_873709.1| 88 Mycoplasma ovipneumoniae SEQ ID NO: 156 184 29 381 420 62 381 420 62 SC01 89 gi|363542550|ref|ZP_09312133.1

If any of the above Cas9 sequences are fused with a peptide or polypeptide at the C-terminus, it is understood that the stop codon will be removed.

Exemplary PAM sequences and their corresponding RKR motifs are provided in Table III.2.

TABLE III.2 Identified PAM sequences and corresponding RKR motifs. RKR PAM sequence motif Strain Name (NA) (AA) Streptococcus pyogenes NGG RKR Streptococcus mutans NGG RKR Streptococcus NGGNG RYR thermophilus A Treponema denticola NAAAAN  VAK Streptococcus NNAAAAW  IYK thermophilus B Campylobacter jejuni NNNNACA NLK Pasteurella multocida GNNNCNNA KDG Neisseria meningitidis NNNNGATT or IGK NNGRRT (R = A or G) Staphylococcus aureus NNGRR NDK (R = A or G)

Exemplary Cas9 core domains are provided in Table III.3.

TABLE III.3 Amino Acid Sequence of Cas9 Core Domains Cas9 Start Cas9 Stop Strain Name (AA pos) (AA pos) Start and Stop numbers refer to the sequence in Table III.1 Staphylococcus aureus 1 772 Streptococcus pyogenes 1 1099 Campulobacter jejuni 1 741

Exemplary PI domains, e.g., altered PI domains, are provided in Tables 111.4 and 111.5.

TABLE III.4 Altered PI Domains PI Start PI Stop Length of PI RKR motif Strain Name (AA pos) (AA POS) (AA) (AA) Start and Stop numbers refer to the sequences in Table III.1 Alicycliphilus denitrificans K601 837 1029 193 --Y Campylobacter jejuni NCTC 11168 741 984 244 -NG Helicobacter mustelae 12198 771 1024 254 -NQ

TABLE III.5 Other Altered PI Domains PI Start PI Stop Length of PI RKR motif Strain Name (AA pos) (AA Pos) (AA) (AA) Start and Stop numbers refer to the sequences in Table III.1 Akkermansia muciniphila ATCC BAA-835 871 1101 231 ALK Ralstonia syzygii R24 821 1062 242 APY Cand. Puniceispirillum marinum IMCC1322 815 1035 221 AYK Fructobacillus fructosus KCTC 3544 1074 1323 250 DGN Eubacterium yurii ATCC 43715 1107 1391 285 DGY Eubacterium dolichum DSM 3991 779 1096 318 DKK Dinoroseobacter shibae DFL 12 851 1079 229 DPI Clostridium cellulolyticum H10 767 1021 255 EGK Pasteurella multocida str. Pm70 815 1056 242 ENN Mycoplasma canis PG 14 907 1233 327 EPK Porphyromonas sp. oral taxon 279 str. F0450 935 1197 263 EPT Filifactor alocis ATCC 35896 1094 1365 272 EVD Aminomonas paucivorans DSM 12260 801 1052 252 EVY Wolinella succinogenes DSM 1740 1034 1409 376 EYK Oenococcus kitaharae DSM 17330 1119 1389 271 GAL CoriobacteriumglomeransPW2 1126 1384 259 GDR Peptoniphilus duerdenii ATCC BAA-1640 1091 1364 274 GDS Bifidobacterium bifidum S17 1138 1420 283 GGL Alicyclobacillus hesperidum URH17-3-68 876 1146 271 GGR Roseburia inulinivorans DSM 16841 895 1152 258 GGT Actinomyces coleocanis DSM 15436 843 1105 263 GKK Odoribacter laneus YIT 12061 1103 1498 396 GKV Coprococcus catus GD-7 1063 1338 276 GNQ Enterococcus faecalis TX0012 829 1150 322 GRK Bacillus smithii 7 3 47FAA 809 1088 280 GSK Legionella pneumophila str. Paris 1021 1372 352 GTM Bacteroides fragilis NCTC 9343 1140 1436 297 IPV Mycoplasma ovipneumoniae SC01 923 1265 343 IRI Actinomyces sp. oral taxon 180 str. F0310 895 1181 287 KEK Treponema sp. JC4 832 1062 231 KIS Fusobacteriumnucleatum ATCC49256 1073 1374 302 KKV Lactobacillus farciminis KCTC 3681 1101 1356 256 KKV Nitratifractor salsuginis DSM 16511 840 1132 293 KMR Lactobacillus coryniformis KCTC 3535 850 1119 270 KNK Mycoplasma mobile 163K 916 1236 321 KNY Flavobacterium branchiophilum FL-15 1182 1473 292 KQK Prevotella timonensis CRIS 5C-B1 957 1218 262 KQQ Methylosinus trichosporium OB3b 830 1082 253 KRP Prevotella sp. C561 1099 1424 326 KRY Mycoplasma gallisepticum str. F 911 1269 359 KTA Lactobacillus rhamnosus GG 1077 1363 287 KYG Wolinella succinogenes DSM 1740 811 1059 249 LPN Streptococcus thermophilus LMD-9 1099 1388 290 MLA Treponema denticola ATCC 35405 1092 1395 304 NDS Bergeyella zoohelcum ATCC 43767 1098 1415 318 NEK Veillonella atypica ACS-134-V-Col7a 1107 1398 292 NGF Neisseria meningitidis Z2491 835 1082 248 NHN Ignavibacterium album JCM 16511 1296 1688 393 NKK Ruminococcus albus 8 853 1156 304 NNF Streptococcus thermophilus LMD-9 811 1121 311 NNK Barnesiella intestinihominis YIT 11860 871 1153 283 NPV Azospirillum sp. B510 911 1168 258 PFH Rhodospirillum rubrum ATCC 11170 863 1173 311 PRG Planococcus antarcticus DSM 14505 1087 1333 247 PYY Staphylococcus pseudintermedius ED99 1073 1334 262 QIV Alcanivorax sp. W11-5 843 1113 271 RIE Bradyrhizobium sp. BTAi1 811 1064 254 RIY Streptococcus pyogenes M1 GAS 1099 1368 270 RKR Streptococcus mutans UA159 1078 1345 268 RKR Streptococcus Pyogenes 1099 1368 270 RKR Bacteroides sp. 20 3 1147 1517 371 RNI S. aureus 772 1053 282 RNK Solobacterium moorei F0204 1062 1327 266 RSG Finegoldia magna ATCC 29328 1081 1348 268 RTE uncultured delta proteobacterium HF0070 07E19 770 1011 242 SGG Acidaminococcus sp. D21 1064 1358 295 SIG Eubacterium rectale ATCC 33656 824 1114 291 SKK Caenispirillum salinarum AK4 1048 1442 395 SLV Acidothermus cellulolyticus 11B 830 1138 309 SPS Catenibacterium mitsuokai DSM 15897 1068 1329 262 SPT Parvibaculum lavamentivorans DS-1 827 1037 211 TGN Staphylococcus lugdunensis M23590 772 1054 283 TKK Streptococcus sanguinis SK49 1123 1421 299 TRM Elusimicrobium minutum Pei191 910 1195 286 TTG Nitrobacter hamburgensis X14 914 1166 253 VAY Mycoplasma synoviae 53 991 1314 324 VGF Sphaerochaeta globus str. Buddy 877 1179 303 VKG Ilyobacter polytropus DSM 2926 837 1092 256 VNG Rhodovulum sp. PH10 821 1059 239 VPY Bifidobacterium longum DJO10A 904 1187 284 VRK

Additional Cas9 molecules are discussed in the section entitled “II. Cas9 Molecules” in International Application WO2015/048577.

Nucleic Acids Encoding Cas9 Molecules Nucleic acids encoding the Cas9 molecules or Cas9 polypeptides, e.g., an eaCas9 molecule or eaCas9 polypeptides, are provided herein.

Exemplary nucleic acids encoding Cas9 molecules are described in Cong et al., SCIENCE 2013, 399(6121): 819-823; Wang et al., CELL 2013, 153(4): 910-918; Mali et al., SCIENCE 2013, 399(6121): 823-826; Jinek et al., SCIENCE 2012, 337(6096): 816-821.

In an embodiment, a nucleic acid encoding a Cas9 molecule, or Cas9 polypeptide, can be a synthetic nucleic acid sequence. For example, the synthetic nucleic acid molecule can be chemically modified, e.g., as described in Section X. In an embodiment, the mRNA, e.g., coding for a Cas9 molecule, or Cas9 polypeptide, disclosed herein, has one or more, e.g., all, of the following properties: it is capped, polyadenylated, substituted with 5-methylcytidine and/or pseudouridine.

In addition, or alternatively, the synthetic nucleic acid sequence can be codon optimized, e.g., at least one non-common codon or less-common codon has been replaced by a codon that is common in the host cell. For example, the synthetic nucleic acid can direct the synthesis of an optimized messenger mRNA, e.g., optimized for expression in a mammalian expression system, e.g., described herein.

In addition, or alternatively, a nucleic acid encoding a Cas9 molecule, or a Cas9 polypeptide, may comprise a nuclear localization sequence (NLS). Nuclear localization sequences are known in the art.

Provided below is an exemplary codon optimized nucleic acid sequence encoding a Cas9 molecule of S. pyogenes.

(SEQ ID NO: 129) ATGGATAAAAAGTACAGCATCGGGCTGGACATCGGTACAAACTCAGTGGG GTGGGCCGTGATTACGGACGAGTACAAGGTACCCTCCAAAAAATTTAAAG TGCTGGGTAACACGGACAGACACTCTATAAAGAAAAATCTTATTGGAGCC TTGCTGTTCGACTCAGGCGAGACAGCCGAAGCCACAAGGTTGAAGCGGAC CGCCAGGAGGCGGTATACCAGGAGAAAGAACCGCATATGCTACCTGCAAG AAATCTTCAGTAACGAGATGGCAAAGGTTGACGATAGCTTTTTCCATCGC CTGGAAGAATCCTTTCTTGTTGAGGAAGACAAGAAGCACGAACGGCACCC CATCTTTGGCAATATTGTCGACGAAGTGGCATATCACGAAAAGTACCCGA CTATCTACCACCTCAGGAAGAAGCTGGTGGACTCTACCGATAAGGCGGAC CTCAGACTTATTTATTTGGCACTCGCCCACATGATTAAATTTAGAGGACA TTTCTTGATCGAGGGCGACCTGAACCCGGACAACAGTGACGTCGATAAGC TGTTCATCCAACTTGTGCAGACCTACAATCAACTGTTCGAAGAAAACCCT ATAAATGCTTCAGGAGTCGACGCTAAAGCAATCCTGTCCGCGCGCCTCTC AAAATCTAGAAGACTTGAGAATCTGATTGCTCAGTTGCCCGGGGAAAAGA AAAATGGATTGTTTGGCAACCTGATCGCCCTCAGTCTCGGACTGACCCCA AATTTCAAAAGTAACTTCGACCTGGCCGAAGACGCTAAGCTCCAGCTGTC CAAGGACACATACGATGACGACCTCGACAATCTGCTGGCCCAGATTGGGG ATCAGTACGCCGATCTCTTTTTGGCAGCAAAGAACCTGTCCGACGCCATC CTGTTGAGCGATATCTTGAGAGTGAACACCGAAATTACTAAAGCACCCCT TAGCGCATCTATGATCAAGCGGTACGACGAGCATCATCAGGATCTGACCC TGCTGAAGGCTCTTGTGAGGCAACAGCTCCCCGAAAAATACAAGGAAATC TTCTTTGACCAGAGCAAAAACGGCTACGCTGGCTATATAGATGGTGGGGC CAGTCAGGAGGAATTCTATAAATTCATCAAGCCCATTCTCGAGAAAATGG ACGGCACAGAGGAGTTGCTGGTCAAACTTAACAGGGAGGACCTGCTGCGG AAGCAGCGGACCTTTGACAACGGGTCTATCCCCCACCAGATTCATCTGGG CGAACTGCACGCAATCCTGAGGAGGCAGGAGGATTTTTATCCTTTTCTTA AAGATAACCGCGAGAAAATAGAAAAGATTCTTACATTCAGGATCCCGTAC TACGTGGGACCTCTCGCCCGGGGCAATTCACGGTTTGCCTGGATGACAAG GAAGTCAGAGGAGACTATTACACCTTGGAACTTCGAAGAAGTGGTGGACA AGGGTGCATCTGCCCAGTCTTTCATCGAGCGGATGACAAATTTTGACAAG AACCTCCCTAATGAGAAGGTGCTGCCCAAACATTCTCTGCTCTACGAGTA CTTTACCGTCTACAATGAACTGACTAAAGTCAAGTACGTCACCGAGGGAA TGAGGAAGCCGGCATTCCTTAGTGGAGAACAGAAGAAGGCGATTGTAGAC CTGTTGTTCAAGACCAACAGGAAGGTGACTGTGAAGCAACTTAAAGAAGA CTACTTTAAGAAGATCGAATGTTTTGACAGTGTGGAAATTTCAGGGGTTG AAGACCGCTTCAATGCGTCATTGGGGACTTACCATGATCTTCTCAAGATC ATAAAGGACAAAGACTTCCTGGACAACGAAGAAAATGAGGATATTCTCGA AGACATCGTCCTCACCCTGACCCTGTTCGAAGACAGGGAAATGATAGAAG AGCGCTTGAAAACCTATGCCCACCTCTTCGACGATAAAGTTATGAAGCAG CTGAAGCGCAGGAGATACACAGGATGGGGAAGATTGTCAAGGAAGCTGAT CAATGGAATTAGGGATAAACAGAGTGGCAAGACCATACTGGATTTCCTCA AATCTGATGGCTTCGCCAATAGGAACTTCATGCAACTGATTCACGATGAC TCTCTTACCTTCAAGGAGGACATTCAAAAGGCTCAGGTGAGCGGGCAGGG AGACTCCCTTCATGAACACATCGCGAATTTGGCAGGTTCCCCCGCTATTA AAAAGGGCATCCTTCAAACTGTCAAGGTGGTGGATGAATTGGTCAAGGTA ATGGGCAGACATAAGCCAGAAAATATTGTGATCGAGATGGCCCGCGAAAA CCAGACCACACAGAAGGGCCAGAAAAATAGTAGAGAGCGGATGAAGAGGA TCGAGGAGGGCATCAAAGAGCTGGGATCTCAGATTCTCAAAGAACACCCC GTAGAAAACACACAGCTGCAGAACGAAAAATTGTACTTGTACTATCTGCA GAACGGCAGAGACATGTACGTCGACCAAGAACTTGATATTAATAGACTGT CCGACTATGACGTAGACCATATCGTGCCCCAGTCCTTCCTGAAGGACGAC TCCATTGATAACAAAGTCTTGACAAGAAGCGACAAGAACAGGGGTAAAAG TGATAATGTGCCTAGCGAGGAGGTGGTGAAAAAAATGAAGAACTACTGGC GACAGCTGCTTAATGCAAAGCTCATTACACAACGGAAGTTCGATAATCTG ACGAAAGCAGAGAGAGGTGGCTTGTCTGAGTTGGACAAGGCAGGGTTTAT TAAGCGGCAGCTGGTGGAAACTAGGCAGATCACAAAGCACGTGGCGCAGA TTTTGGACAGCCGGATGAACACAAAATACGACGAAAATGATAAACTGATA CGAGAGGTCAAAGTTATCACGCTGAAAAGCAAGCTGGTGTCCGATTTTCG GAAAGACTTCCAGTTCTACAAAGTTCGCGAGATTAATAACTACCATCATG CTCACGATGCGTACCTGAACGCTGTTGTCGGGACCGCCTTGATAAAGAAG TACCCAAAGCTGGAATCCGAGTTCGTATACGGGGATTACAAAGTGTACGA TGTGAGGAAAATGATAGCCAAGTCCGAGCAGGAGATTGGAAAGGCCACAG CTAAGTACTTCTTTTATTCTAACATCATGAATTTTTTTAAGACGGAAATT ACCCTGGCCAACGGAGAGATCAGAAAGCGGCCCCTTATAGAGACAAATGG TGAAACAGGTGAAATCGTCTGGGATAAGGGCAGGGATTTCGCTACTGTGA GGAAGGTGCTGAGTATGCCACAGGTAAATATCGTGAAAAAAACCGAAGTA CAGACCGGAGGATTTTCCAAGGAAAGCATTTTGCCTAAAAGAAACTCAGA CAAGCTCATCGCCCGCAAGAAAGATTGGGACCCTAAGAAATACGGGGGAT TTGACTCACCCACCGTAGCCTATTCTGTGCTGGTGGTAGCTAAGGTGGAA AAAGGAAAGTCTAAGAAGCTGAAGTCCGTGAAGGAACTCTTGGGAATCAC TATCATGGAAAGATCATCCTTTGAAAAGAACCCTATCGATTTCCTGGAGG CTAAGGGTTACAAGGAGGTCAAGAAAGACCTCATCATTAAACTGCCAAAA TACTCTCTCTTCGAGCTGGAAAATGGCAGGAAGAGAATGTTGGCCAGCGC CGGAGAGCTGCAAAAGGGAAACGAGCTTGCTCTGCCCTCCAAATATGTTA ATTTTCTCTATCTCGCTTCCCACTATGAAAAGCTGAAAGGGTCTCCCGAA GATAACGAGCAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTATCTGGA TGAAATAATCGAACAAATAAGCGAGTTCAGCAAAAGGGTTATCCTGGCGG ATGCTAATTTGGACAAAGTACTGTCTGCTTATAACAAGCACCGGGATAAG CCTATTAGGGAACAAGCCGAGAATATAATTCACCTCTTTACACTCACGAA TCTCGGAGCCCCCGCCGCCTTCAAATACTTTGATACGACTATCGACCGGA AACGGTATACCAGTACCAAAGAGGTCCTCGATGCCACCCTCATCCACCAG TCAATTACTGGCCTGTACGAAACACGGATCGACCTCTCTCAACTGGGCGG CGACTAG

Provided below is the corresponding amino acid sequence of a S. pyogenes Cas9 molecule.

(SEQ ID NO: 130) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGD*

Provided below is an exemplary codon optimized nucleic acid sequence encoding a Cas9 molecule of N. meningitidis.

(SEQ ID NO: 131) ATGGCCGCCTTCAAGCCCAACCCCATCAACTACATCCTGGGCCTGGACAT CGGCATCGCCAGCGTGGGCTGGGCCATGGTGGAGATCGACGAGGACGAGA ACCCCATCTGCCTGATCGACCTGGGTGTGCGCGTGTTCGAGCGCGCTGAG GTGCCCAAGACTGGTGACAGTCTGGCTATGGCTCGCCGGCTTGCTCGCTC TGTTCGGCGCCTTACTCGCCGGCGCGCTCACCGCCTTCTGCGCGCTCGCC GCCTGCTGAAGCGCGAGGGTGTGCTGCAGGCTGCCGACTTCGACGAGAAC GGCCTGATCAAGAGCCTGCCCAACACTCCTTGGCAGCTGCGCGCTGCCGC TCTGGACCGCAAGCTGACTCCTCTGGAGTGGAGCGCCGTGCTGCTGCACC TGATCAAGCACCGCGGCTACCTGAGCCAGCGCAAGAACGAGGGCGAGACC GCCGACAAGGAGCTGGGTGCTCTGCTGAAGGGCGTGGCCGACAACGCCCA CGCCCTGCAGACTGGTGACTTCCGCACTCCTGCTGAGCTGGCCCTGAACA AGTTCGAGAAGGAGAGCGGCCACATCCGCAACCAGCGCGGCGACTACAGC CACACCTTCAGCCGCAAGGACCTGCAGGCCGAGCTGATCCTGCTGTTCGA GAAGCAGAAGGAGTTCGGCAACCCCCACGTGAGCGGCGGCCTGAAGGAGG GCATCGAGACCCTGCTGATGACCCAGCGCCCCGCCCTGAGCGGCGACGCC GTGCAGAAGATGCTGGGCCACTGCACCTTCGAGCCAGCCGAGCCCAAGGC CGCCAAGAACACCTACACCGCCGAGCGCTTCATCTGGCTGACCAAGCTGA ACAACCTGCGCATCCTGGAGCAGGGCAGCGAGCGCCCCCTGACCGACACC GAGCGCGCCACCCTGATGGACGAGCCCTACCGCAAGAGCAAGCTGACCTA CGCCCAGGCCCGCAAGCTGCTGGGTCTGGAGGACACCGCCTTCTTCAAGG GCCTGCGCTACGGCAAGGACAACGCCGAGGCCAGCACCCTGATGGAGATG AAGGCCTACCACGCCATCAGCCGCGCCCTGGAGAAGGAGGGCCTGAAGGA CAAGAAGAGTCCTCTGAACCTGAGCCCCGAGCTGCAGGACGAGATCGGCA CCGCCTTCAGCCTGTTCAAGACCGACGAGGACATCACCGGCCGCCTGAAG GACCGCATCCAGCCCGAGATCCTGGAGGCCCTGCTGAAGCACATCAGCTT CGACAAGTTCGTGCAGATCAGCCTGAAGGCCCTGCGCCGCATCGTGCCCC TGATGGAGCAGGGCAAGCGCTACGACGAGGCCTGCGCCGAGATCTACGGC GACCACTACGGCAAGAAGAACACCGAGGAGAAGATCTACCTGCCTCCTAT CCCCGCCGACGAGATCCGCAACCCCGTGGTGCTGCGCGCCCTGAGCCAGG CCCGCAAGGTGATCAACGGCGTGGTGCGCCGCTACGGCAGCCCCGCCCGC ATCCACATCGAGACCGCCCGCGAGGTGGGCAAGAGCTTCAAGGACCGCAA GGAGATCGAGAAGCGCCAGGAGGAGAACCGCAAGGACCGCGAGAAGGCCG CCGCCAAGTTCCGCGAGTACTTCCCCAACTTCGTGGGCGAGCCCAAGAGC AAGGACATCCTGAAGCTGCGCCTGTACGAGCAGCAGCACGGCAAGTGCCT GTACAGCGGCAAGGAGATCAACCTGGGCCGCCTGAACGAGAAGGGCTACG TGGAGATCGACCACGCCCTGCCCTTCAGCCGCACCTGGGACGACAGCTTC AACAACAAGGTGCTGGTGCTGGGCAGCGAGAACCAGAACAAGGGCAACCA GACCCCCTACGAGTACTTCAACGGCAAGGACAACAGCCGCGAGTGGCAGG AGTTCAAGGCCCGCGTGGAGACCAGCCGCTTCCCCCGCAGCAAGAAGCAG CGCATCCTGCTGCAGAAGTTCGACGAGGACGGCTTCAAGGAGCGCAACCT GAACGACACCCGCTACGTGAACCGCTTCCTGTGCCAGTTCGTGGCCGACC GCATGCGCCTGACCGGCAAGGGCAAGAAGCGCGTGTTCGCCAGCAACGGC CAGATCACCAACCTGCTGCGCGGCTTCTGGGGCCTGCGCAAGGTGCGCGC CGAGAACGACCGCCACCACGCCCTGGACGCCGTGGTGGTGGCCTGCAGCA CCGTGGCCATGCAGCAGAAGATCACCCGCTTCGTGCGCTACAAGGAGATG AACGCCTTCGACGGTAAAACCATCGACAAGGAGACCGGCGAGGTGCTGCA CCAGAAGACCCACTTCCCCCAGCCCTGGGAGTTCTTCGCCCAGGAGGTGA TGATCCGCGTGTTCGGCAAGCCCGACGGCAAGCCCGAGTTCGAGGAGGCC GACACCCCCGAGAAGCTGCGCACCCTGCTGGCCGAGAAGCTGAGCAGCCG CCCTGAGGCCGTGCACGAGTACGTGACTCCTCTGTTCGTGAGCCGCGCCC CCAACCGCAAGATGAGCGGTCAGGGTCACATGGAGACCGTGAAGAGCGCC AAGCGCCTGGACGAGGGCGTGAGCGTGCTGCGCGTGCCCCTGACCCAGCT GAAGCTGAAGGACCTGGAGAAGATGGTGAACCGCGAGCGCGAGCCCAAGC TGTACGAGGCCCTGAAGGCCCGCCTGGAGGCCCACAAGGACGACCCCGCC AAGGCCTTCGCCGAGCCCTTCTACAAGTACGACAAGGCCGGCAACCGCAC CCAGCAGGTGAAGGCCGTGCGCGTGGAGCAGGTGCAGAAGACCGGCGTGT GGGTGCGCAACCACAACGGCATCGCCGACAACGCCACCATGGTGCGCGTG GACGTGTTCGAGAAGGGCGACAAGTACTACCTGGTGCCCATCTACAGCTG GCAGGTGGCCAAGGGCATCCTGCCCGACCGCGCCGTGGTGCAGGGCAAGG ACGAGGAGGACTGGCAGCTGATCGACGACAGCTTCAACTTCAAGTTCAGC CTGCACCCCAACGACCTGGTGGAGGTGATCACCAAGAAGGCCCGCATGTT CGGCTACTTCGCCAGCTGCCACCGCGGCACCGGCAACATCAACATCCGCA TCCACGACCTGGACCACAAGATCGGCAAGAACGGCATCCTGGAGGGCATC GGCGTGAAGACCGCCCTGAGCTTCCAGAAGTACCAGATCGACGAGCTGGG CAAGGAGATCCGCCCCTGCCGCCTGAAGAAGCGCCCTCCTGTGCGCTAA

Provided below is the corresponding amino acid sequence of a N. meningitidis Cas9 molecule.

(SEQ ID NO: 132) MAAFKPNPINYILGLDIGIASVGWAMVEIDEDENPICLIDLGVRVFERAE VPKTGDSLAMARRLARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDEN GLIKSLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGET ADKELGALLKGVADNAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYS HTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLMTQRPALSGDA VQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDT ERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEM KAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLK DRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYG DHYGKKNTEEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPAR IHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFREYFPNFVGEPKS KDILKLRLYEQQHGKCLYSGKEINLGRLNEKGYVEIDHALPFSRTWDDSF NNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQ RILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRVFASNG QITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEM NAFDGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEA DTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSA KRLDEGVSVLRVPLTQLKLKDLEKMVNREREPKLYEALKARLEAHKDDPA KAFAEPFYKYDKAGNRTQQVKAVRVEQVQKTGVWVRNHNGIADNATMVRV DVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQLIDDSFNFKFS LHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILEGI GVKTALSFQKYQIDELGKEIRPCRLKKRPPVR*

Provided below is an amino acid sequence of a S. aureus Cas9 molecule.

(SEQ ID NO: 133) MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSK RGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKL SEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYV AELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDT YIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYA YNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIA KEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQ IAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVV KRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQ TNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNP FNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKIS YETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTR YATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKH HAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEY KEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTL IVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDE KNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNS RNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEA KKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDIT YREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQII KKG*

Provided below is an exemplary codon optimized nucleic acid sequence encoding a S. aureus Cas9 molecule.

(SEQ ID NO: 134) ATGAAAAGGAACTACATTCTGGGGCTGGACATCGGGATTACAAGCGTGGG GTATGGGATTATTGACTATGAAACAAGGGACGTGATCGACGCAGGCGTCA GACTGTTCAAGGAGGCCAACGTGGAAAACAATGAGGGACGGAGAAGCAAG AGGGGAGCCAGGCGCCTGAAACGACGGAGAAGGCACAGAATCCAGAGGGT GAAGAAACTGCTGTTCGATTACAACCTGCTGACCGACCATTCTGAGCTGA GTGGAATTAATCCTTATGAAGCCAGGGTGAAAGGCCTGAGTCAGAAGCTG TCAGAGGAAGAGTTTTCCGCAGCTCTGCTGCACCTGGCTAAGCGCCGAGG AGTGCATAACGTCAATGAGGTGGAAGAGGACACCGGCAACGAGCTGTCTA CAAAGGAACAGATCTCACGCAATAGCAAAGCTCTGGAAGAGAAGTATGTC GCAGAGCTGCAGCTGGAACGGCTGAAGAAAGATGGCGAGGTGAGAGGGTC AATTAATAGGTTCAAGACAAGCGACTACGTCAAAGAAGCCAAGCAGCTGC TGAAAGTGCAGAAGGCTTACCACCAGCTGGATCAGAGCTTCATCGATACT TATATCGACCTGCTGGAGACTCGGAGAACCTACTATGAGGGACCAGGAGA AGGGAGCCCCTTCGGATGGAAAGACATCAAGGAATGGTACGAGATGCTGA TGGGACATTGCACCTATTTTCCAGAAGAGCTGAGAAGCGTCAAGTACGCT TATAACGCAGATCTGTACAACGCCCTGAATGACCTGAACAACCTGGTCAT CACCAGGGATGAAAACGAGAAACTGGAATACTATGAGAAGTTCCAGATCA TCGAAAACGTGTTTAAGCAGAAGAAAAAGCCTACACTGAAACAGATTGCT AAGGAGATCCTGGTCAACGAAGAGGACATCAAGGGCTACCGGGTGACAAG CACTGGAAAACCAGAGTTCACCAATCTGAAAGTGTATCACGATATTAAGG ACATCACAGCACGGAAAGAAATCATTGAGAACGCCGAACTGCTGGATCAG ATTGCTAAGATCCTGACTATCTACCAGAGCTCCGAGGACATCCAGGAAGA GCTGACTAACCTGAACAGCGAGCTGACCCAGGAAGAGATCGAACAGATTA GTAATCTGAAGGGGTACACCGGAACACACAACCTGTCCCTGAAAGCTATC AATCTGATTCTGGATGAGCTGTGGCATACAAACGACAATCAGATTGCAAT CTTTAACCGGCTGAAGCTGGTCCCAAAAAAGGTGGACCTGAGTCAGCAGA AAGAGATCCCAACCACACTGGTGGACGATTTCATTCTGTCACCCGTGGTC AAGCGGAGCTTCATCCAGAGCATCAAAGTGATCAACGCCATCATCAAGAA GTACGGCCTGCCCAATGATATCATTATCGAGCTGGCTAGGGAGAAGAACA GCAAGGACGCACAGAAGATGATCAATGAGATGCAGAAACGAAACCGGCAG ACCAATGAACGCATTGAAGAGATTATCCGAACTACCGGGAAAGAGAACGC AAAGTACCTGATTGAAAAAATCAAGCTGCACGATATGCAGGAGGGAAAGT GTCTGTATTCTCTGGAGGCCATCCCCCTGGAGGACCTGCTGAACAATCCA TTCAACTACGAGGTCGATCATATTATCCCCAGAAGCGTGTCCTTCGACAA TTCCTTTAACAACAAGGTGCTGGTCAAGCAGGAAGAGAACTCTAAAAAGG GCAATAGGACTCCTTTCCAGTACCTGTCTAGTTCAGATTCCAAGATCTCT TACGAAACCTTTAAAAAGCACATTCTGAATCTGGCCAAAGGAAAGGGCCG CATCAGCAAGACCAAAAAGGAGTACCTGCTGGAAGAGCGGGACATCAACA GATTCTCCGTCCAGAAGGATTTTATTAACCGGAATCTGGTGGACACAAGA TACGCTACTCGCGGCCTGATGAATCTGCTGCGATCCTATTTCCGGGTGAA CAATCTGGATGTGAAAGTCAAGTCCATCAACGGCGGGTTCACATCTTTTC TGAGGCGCAAATGGAAGTTTAAAAAGGAGCGCAACAAAGGGTACAAGCAC CATGCCGAAGATGCTCTGATTATCGCAAATGCCGACTTCATCTTTAAGGA GTGGAAAAAGCTGGACAAAGCCAAGAAAGTGATGGAGAACCAGATGTTCG AAGAGAAGCAGGCCGAATCTATGCCCGAAATCGAGACAGAACAGGAGTAC AAGGAGATTTTCATCACTCCTCACCAGATCAAGCATATCAAGGATTTCAA GGACTACAAGTACTCTCACCGGGTGGATAAAAAGCCCAACAGAGAGCTGA TCAATGACACCCTGTATAGTACAAGAAAAGACGATAAGGGGAATACCCTG ATTGTGAACAATCTGAACGGACTGTACGACAAAGATAATGACAAGCTGAA AAAGCTGATCAACAAAAGTCCCGAGAAGCTGCTGATGTACCACCATGATC CTCAGACATATCAGAAACTGAAGCTGATTATGGAGCAGTACGGCGACGAG AAGAACCCACTGTATAAGTACTATGAAGAGACTGGGAACTACCTGACCAA GTATAGCAAAAAGGATAATGGCCCCGTGATCAAGAAGATCAAGTACTATG GGAACAAGCTGAATGCCCATCTGGACATCACAGACGATTACCCTAACAGT CGCAACAAGGTGGTCAAGCTGTCACTGAAGCCATACAGATTCGATGTCTA TCTGGACAACGGCGTGTATAAATTTGTGACTGTCAAGAATCTGGATGTCA TCAAAAAGGAGAACTACTATGAAGTGAATAGCAAGTGCTACGAAGAGGCT AAAAAGCTGAAAAAGATTAGCAACCAGGCAGAGTTCATCGCCTCCTTTTA CAACAACGACCTGATTAAGATCAATGGCGAACTGTATAGGGTCATCGGGG TGAACAATGATCTGCTGAACCGCATTGAAGTGAATATGATTGACATCACT TACCGAGAGTATCTGGAAAACATGAATGATAAGCGCCCCCCTCGAATTAT CAAAACAATTGCCTCTAAGACTCAGAGTATCAAAAAGTACTCAACCGACA TTCTGGGAAACCTGTATGAGGTGAAGAGCAAAAAGCACCCTCAGATTATC AAAAAGGGC

If any of the above Cas9 sequences are fused with a peptide or polypeptide at the C-terminus, it is understood that the stop codon will be removed.

Other Cas Molecules and Cas Polypeptides

Various types of Cas molecules or Cas polypeptides can be used to practice the inventions disclosed herein. In some embodiments, Cas molecules of Type II Cas systems are used. In other embodiments, Cas molecules of other Cas systems are used. For example, Type I or Type III Cas molecules may be used. Exemplary Cas molecules (and Cas systems) are described, e.g., in Haft et al. (2005) PLOS COMPUTATIONAL BIOLOGY 1(6): e60, and in Makarova et al. (2011) NATURE REVIEW MICROBIOLOGY 9: 467-477, the contents of which are incorporated herein by reference in their entirety. Exemplary Cas molecules (and Cas systems) are also shown in Table III.6.

TABLE III.6 Cas Systems Families Structure of (and superfamily) Gene System type Name from encoded protein of encoded name‡ or subtype Haft et al.§ (PDB accessions)¶ protein#** Representatives cas1 Type I cas1 3GOD, 3LFX and COG1518 SERP2463, SPy1047 Type II 2YZS and ygbT Type III cas2 Type I cas2 2IVY, 2I8E and COG1343 and SERP2462, SPy1048, Type II 3EXC COG3512 SPy1723 (N-terminal Type III domain) and ygbF cas3′ Type I‡‡ cas3 NA COG1203 APE1232 and ygcB cas3″ Subtype I-A NA NA COG2254 APE1231 and Subtype I-B BH0336 cas4 Subtype I-A cas4 and csa1 NA COG1468 APE1239 and Subtype I-B BH0340 Subtype I-C Subtype I-D Subtype II-B cas5 Subtype I-A cas5a, cas5d, 3KG4 COG1688 APE1234, BH0337, Subtype I-B cas5e, cas5h, (RAMP) devS and ygcI Subtype I-C cas5p, cas5t Subtype I-E and cmx5 cas6 Subtype I-A cas6 and cmx6 3I4H COG1583 and PF1131 and slr7014 Subtype I-B COG5551 Subtype I-D (RAMP) Subtype III-A Subtype III-B cas6e Subtype I-E cse3 1WJ9 (RAMP) ygcH cas6f Subtype I-F csy4 2XLJ (RAMP) y1727 cas7 Subtype I-A csa2, csd2, NA COG1857 and devR and ygcJ Subtype I-B cse4, csh2, COG3649 Subtype I-C csp1 and cst2 (RAMP) Subtype I-E cas8a1 Subtype I-A‡‡ cmx1, cst1, NA BH0338-like LA3191§§ and csx8, csx13 PG2018§§ and CXXC- CXXC cas8a2 Subtype I-A‡‡ csa4 and csx9 NA PH0918 AF0070, AF1873, MJ0385, PF0637, PH0918and SSO1401 cas8b Subtype I-B‡‡ csh1 and NA BH0338-like MTH1090 and TM1802 TM1802 cas8c Subtype I-C‡‡ csd1 and csp2 NA BH0338-like BH0338 cas9 Type II‡‡ csn1 and csx12 NA COG3513 FTN_0757 and SPy1046 cas10 Type III‡‡ cmr2, csm1 NA COG1353 MTH326, Rv2823c§§ and csx11 and TM1794§§ cas10d Subtype I-D‡‡ csc3 NA COG1353 slr7011 csy1 Subtype I-F‡‡ csy1 NA y1724-like y1724 csy2 Subtype I-F csy2 NA (RAMP) y1725 csy3 Subtype I-F csy3 NA (RAMP) y1726 cse1 Subtype I-E‡‡ cse1 NA YgcL-like ygcL cse2 Subtype I-E cse2 2ZCA YgcK-like ygcK csc1 Subtype I-D csc1 NA alr1563-like alr1563 (RAMP) csc2 Subtype I-D csc1 and csc2 NA COG1337 slr7012 (RAMP) csa5 Subtype I-A csa5 NA AF1870 AF1870, MJ0380, PF0643 and SSO1398 csn2 Subtype II-A csn2 NA SPy1049-like SPy1049 csm2 Subtype III-A‡‡ csm2 NA COG1421 MTH1081 and SERP2460 csm3 Subtype III-A csc2 and NA COG1337 MTH1080 and csm3 (RAMP) SERP2459 csm4 Subtype III-A csm4 NA COG1567 MTH1079 and (RAMP) SERP2458 csm5 Subtype III-A csm5 NA COG1332 MTH1078 and (RAMP) SERP2457 csm6 Subtype III-A APE2256 2WTE COG1517 APE2256 and and csm6 SSO1445 cmr1 Subtype III-B cmr1 NA COG1367 PF1130 (RAMP) cmr3 Subtype III-B cmr3 NA COG1769 PF1128 (RAMP) cmr4 Subtype III-B cmr4 NA COG1336 PF1126 (RAMP) cmr5 Subtype III-B‡‡ cmr5 2ZOP and 2OEB COG3337 MTH324 and PF1125 cmr6 Subtype III-B cmr6 NA COG1604 PF1124 (RAMP) csb1 Subtype I-U GSU0053 NA (RAMP) Balac_1306 and GSU0053 csb2 Subtype I-U§§ NA NA (RAMP) Balac_1305 and GSU0054 csb3 Subtype I-U NA NA (RAMP) Balac_1303§§ csx17 Subtype I-U NA NA NA Btus_2683 csx14 Subtype l-U NA NA NA GSU0052 csx10 Subtype I-U csx10 NA (RAMP) Caur_2274 csx16 Subtype III-U VVA1548 NA NA VVA1548 csaX Subtype III-U csaX NA NA SSO1438 csx3 Subtype III-U csx3 NA NA AF1864 csx1 Subtype III-U csa3, csx1, 1XMX and 2171 COG1517 and MJ1666, NE0113, csx2, DXTHG, COG4006 PF1127 and TM1812 NE0113 and TIGR02710 csx15 Unknown NA NA TTE2665 TTE2665 csf1 Type U csf1 NA NA AFE_1038 csf2 Type U csf2 NA (RAMP) AFE_1039 csf3 Type U csf3 NA (RAMP) AFE_1040 csf4 Type U csf4 NA NA AFE_1037

Linkers

In one aspect, the Cas9 molecules described herein comprise a REC2 deletion, a REC1_(CT) deletion, and/or a REC1_(SUB) deletion, and a linker disposed between the amino acid residues that flank each deletion, e.g., between the amino acid residues N-terminal and C-terminal to the deletion. Preferably, the linkers used between the amino acid residues that flank each deletion include properties such that the resulting Cas9 molecule properly folds and possesses functional activity. In an embodiment, Cas9 molecules described herein do not contain a linker between the amino acid residues that flank a deletion.

In another aspect, the Cas9 molecule described herein comprises a linker disposed between a Cas9 core domain (comprising a REC2 deletion, a REC1_(CT) deletion, and/or a REC1_(SUB) deletion) and a heterologous PI domain. The linker is disposed between the C-terminus of the Cas9 core domain and the N-terminus of a heterologous PI domain. In some embodiments, the Cas9 molecule does not contain a linker disposed between a Cas9 core domain and a heterologous PI domain, e.g., the C-terminus of the Cas9 core domain is linked to the N-terminus of the heterologous PI domain.

Linkers for use in generating recombinant proteins, e.g., multi-domain proteins, are known in the art (Chen et al., Adv Drug Delivery Rev, 65:1357-69, 2013). As an example. any linkers known in the art that maintain the conformation or native fold of the Cas9 molecule (thereby retaining Cas9 activity) can be used. Several properties of linkers, such as length, hydrophobilicty, intrinsic properties of the amino acids residues themselves, and secondary structure should be considered in the context of the goal to maintain native conformation and functional activity of Cas9.

A flexible linker can be utilized in the Cas9 molecules described herein. Flexible linkers allow a certain degree of movement and/or interaction within and between the joined domains or regions of the protein. Generally, flexible linkers are composed of small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The small size of these amino acids provides flexibility and allows mobility of the connected domains or regions. Furthermore, the incorporation of Ser or Thr can help maintain the stability of the linker in aqueous solutions by hydrogen bonding with the water molecules, thereby reducing unfavorable interactions between the linker and the other protein moieties. Commonly used flexible linkers are comprised of sequences that primarily consist of Gly and Ser residues. Often, these flexible linkers consist of repeating units of a combination of Gly and Ser residues, e.g., (GGS)x, where the number of repeating units, e.g., x, can be optimized to achieve the appropriate separation of other domains or regions of the protein.

In some cases, a rigid linker may be preferred if there is significant distance between the joined domains or regions, or to maintain a fixed distance between the joined domains or regions of a protein and independent functions of the domains/regions. Rigid linkers often have defined secondary structure, e.g., alpha helix, or other stabilizing interactions, e.g., salt bridges and disulfide bonds. Rigid linkers commonly contain multiple Pro residues, or repeating combinations of Glu-Pro or Lys-Pro because Pro imposes a strong conformation constraint due to its structure.

In an embodiment, the linker comprises:

-   -   1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 amino acid residues;     -   Less than 10, 20 or 30 amino acid residues;     -   (GGS)x, wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID         NO: 339);     -   (GS)x, wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO:         341); or     -   (SGSETPGTSESATPES)x, where x is 1, 2, 3, or 4 (also referred to         herein interchangeably as XTEN or the XTEN linker) (SEQ ID NO:         344). Alternative linkers may include (GSAGSAAGSGEF)_(x),         wherein x is 1, 2, 3 or 4 (SEQ ID NO: 345) and         (SIVAQLSRPDPA)_(x), wherein x is 1, 2, 3 or 4 (SEQ ID NO: 346).         In some embodiments, a combination of linkers may be used, e.g.,         a combination of an XTEN linker (or other alternative linker), a         (GS)_(x) linker, and/or a (GGS)_(x) linker.

In some embodiments, specific linkers may be preferred depending on the specific deleted sequence or region or the heterologous PI domain. Examples of preferred linkers for Cas9 with a REC2 deletion, a REC1_(CT) deletion, or a REC1_(SUB) deletion are provided in further detail below.

In some embodiments, the linker length is from about 6 to 60 amino acids. The linker may be, e.g., 6-10, 10-15, 15-20, 20-30, 30-40, 40-50, or 50-60 amino acids in length. The linker may be, e.g., at least 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 amino acids in length. In other embodiments, the linker is, e.g., at most 7, 8, 9, 10, 15, 20, 30, 40, 50, or 60 amino acids in length. Ranges comprising any combination of these endpoints are also envisioned.

In some embodiments, the linker is encoded by a nucleic acid sequence of 6 to 60 nucleotides or base pairs. The nucleic acid may be, e.g., 6-10, 10-15, 15-20, 20-30, 30-40, 40-50, or 50-60 nucleotides in length. The linker may be, e.g., at least 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 nucleotides in length. In some embodiments, the linker is, e.g., at most 7, 8, 9, 10, 15, 20, 30, 40, 50, or 60 nucleotides in length. Ranges comprising any combination of these endpoints are also envisioned.

In some embodiments, the linker comprises glycine and serine residues. In some embodiments the linker consists of glycine and serine residues. For instance, the linker may comprise one of more modules such as GGS, GSGS (SEQ ID NO: 128), GGGS (SEQ ID NO: 139), GGGGS (SEQ ID NO: 140) or GGSG (SEQ ID NO: 141). In some embodiments, the linker comprises a plurality of modules comprising glycine and serine, e.g., at least 2, 3, 4, 5, 10, or 15 of these modules, and/or at most 3, 4, 5, 10, 15, or 20 of these modules, or any combination of these endpoints. In some embodiments, each module in the linker has the same sequence, and in other embodiments, at least two modules in a linker have different sequences from each other.

In some embodiments, the linker is an XTEN linker or a variation of an XTEN linker such as SGSETPGTSESA (SEQ ID NO: 135), SGSETPGTSESATPES (SEQ ID NO: 136), or SGSETPGTSESATPEGGSGGS (SEQ ID NO: 137). Additional information on the XTEN linker may be found in Schellenberger et al. (2009), NATURE BIOTECHNOLOGY 27: 1186-1190, the entire contents of which are incorporated herein by reference. Exemplary linker modules are given in Table IV.

TABLE III.7 Exemplary Linker Modules GGS GSGS (SEQ ID NO: 138) GGGS (SEQ ID NO: 139) GGGGS (SEQ ID NO: 140) GGSG (SEQ ID NO: 141) SGSETPGTSESA (SEQ ID NO: 135) SGSETPGTSESATPES (SEQ ID NO: 136) SGSETPGTSESATPEGGSGGS (SEQ ID NO: 137)

Additional exemplary linker modules are given in Table V.

TABLE III.8 Exemplary Linker Modules Length Name Description (nt) BBa_J176131 PLrigid 60 BBa_J18920 2aa GS linker 6 BBa_J18921 6aa [GS]x linker (SEQ ID NO: 142) 18 BBa_J18922 10aa [GS]x linker (SEQ ID NO: 143) 30 BBa_K105012 10 aa flexible protein domain linker 30 BBa_K133132 8 aa protein domain linker 24 BBa_K1486003 flexible linker 2x (GGGS) (SEQ ID NO: 139) 24 BBa_K1486004 flexible linker 2x (GGGGS) (SEQ ID NO: 140) 30 BBa_K1486037 linker 39 BBa_K157009 Split fluorophore linker; Freiburg standard 51 BBa_K157013 15 aa flexible glycine-serine protein 45 domain linker; Freiburg standard BBa_K243004 Short Linker (Gly-Gly-Ser-Gly) (SEQ ID NO: 141) 12 BBa_K243005 Middle Linker (Gly-Gly-Ser-Gly)x2 (SEQ ID NO: 144) 24 BBa_K243006 Long Linker (Gly-Gly-Ser-Gly)x3 (SEQ ID NO: 145) 36 BBa_K243029 GSAT Linker 108 BBa_K243030 SEG 108 BBa_K404300 SEG-Linker 108 BBa_K404301 GSAT-Linker 108 BBa_K404303 Z-EGFR-1907_Short-Linker 192 BBa_K404304 Z-EGFR-1907_Middle-Linker 204 BBa_K404305 Z-EGFR-1907_Long-Linker 216 BBa_K404306 Z-EGFR-1907_SEG-Linker 288 BBa_K416001 (Gly4Ser)3 Flexible Peptide Linker (SEQ ID NO: 146) 45 BBa_K648005 Short Fusion Protein Linker: GGSG with 12 standard 25 prefix/suffix  (SEQ ID NO: 141) BBa_K648006 Long 10AA Fusion Protein Linker with 30 Standard 25 Prefix/Suffix BBa_K648007 Medium 6AA Fusion Protein Linker: 18 GGSGGS with Standard 25 Prefix/Suffix  (SEQ ID NO: 147)

Linkers can comprise a direct bond or an atom such as, e.g., an oxygen (O) or sulfur (S), a unit such as —NR— wherein R is hydrogen or alkyl, —C(O)—, —C(O)O—, —C(O)NH—, SO, SO₂, —SO₂NH— or a chain of atoms, such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, heteroarylalkyl. In some embodiments, one or more methylenes in the chain of atoms can be replaced with one or more of O, S, S(O), SO₂, —SO₂NH—, —NR—, —NR₂, —C(O)—, —C(O)O—, —C(O)NH—, a cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocyclic.

In some embodiments, the linker comprises an alkylene moiety or a heteroalkylene moiety (e.g., an alkylene glycol moiety such as ethylene glycol). In some embodiments, a linker comprises a poly-L-glutamic acid, polylactic acid, poly(ethyleneimine), an oligosaccharide, an amino acid (e.g., glycine), an amino acid chain, or any other suitable linkage. The linker groups can be biologically inactive, such as a PEG, polyglycolic acid, or polylactic acid chain. In certain embodiments, the linker group represents a derivatized or non-derivatized amino acid (e.g., glycine).

The length of the linkers can be easily adjusted by changing the copy number of repeating units to achieve an optimal distance between the domains or regions that are to be joined. In embodiments, the different linkers can be joined together to achieve optimal distance, flexibility, or rigidity between the joined domains or regions of a Cas9 molecule.

IV. Functional Analysis of Candidate Molecules

Candidate Cas9 molecules, candidate gRNA molecules, candidate Cas9 molecule/gRNA molecule complexes, can be evaluated by art-known methods or as described herein. For example, exemplary methods for evaluating the endonuclease activity of Cas9 molecule are described, e.g., in Jinek et al. (2012) SCIENCE 337(6096): 816-821. The methods in this section may be used, e.g., to test various portions of a gRNA, for example, the targeting domain, the first complementarity domain, the linking domain, the second complementarity domain, the proximal domain, or the tail domain. In some embodiments, the methods in this section are tested to determine whether modifications made in one or more of these domains interfere with targeting efficacy. A gRNA with a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in a system of this section.

Binding and Cleavage Assay: Testing the Endonuclease Activity of Cas9 Molecule

The ability of a Cas9 molecule/gRNA molecule complex to bind to and cleave a target nucleic acid can be evaluated in a plasmid cleavage assay. In this assay, synthetic or in vitro-transcribed gRNA molecule is pre-annealed prior to the reaction by heating to 95° C. and slowly cooling down to room temperature. Native or restriction digest-linearized plasmid DNA (300 ng (˜8 nM)) is incubated for 60 min at 37° C. with purified Cas9 protein molecule (50-500 nM) and gRNA (50-500 nM, 1:1) in a Cas9 plasmid cleavage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 0.5 mM DTT, 0.1 mM EDTA) with or without 10 mM MgCl₂. The reactions are stopped with 5×DNA loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA), resolved by a 0.8 or 1% agarose gel electrophoresis and visualized by ethidium bromide staining. The resulting cleavage products indicate whether the Cas9 molecule cleaves both DNA strands, or only one of the two strands. For example, linear DNA products indicate the cleavage of both DNA strands. Nicked open circular products indicate that only one of the two strands is cleaved.

Alternatively, the ability of a Cas9 molecule/gRNA molecule complex to bind to and cleave a target nucleic acid can be evaluated in an oligonucleotide DNA cleavage assay. In this assay, DNA oligonucleotides (10 pmol) are radiolabeled by incubating with 5 units T4 polynucleotide kinase and ˜3-6 pmol (˜20-40 mCi) [γ-32P]-ATP in 1× T4 polynucleotide kinase reaction buffer at 37° C. for 30 min, in a 50 μL reaction. After heat inactivation (65° C. for 20 min), reactions are purified through a column to remove unincorporated label. Duplex substrates (100 nM) are generated by annealing labeled oligonucleotides with equimolar amounts of unlabeled complementary oligonucleotide at 95° C. for 3 min, followed by slow cooling to room temperature. For cleavage assays, gRNA molecules are annealed by heating to 95° C. for 30 s, followed by slow cooling to room temperature. Cas9 (500 nM final concentration) is pre-incubated with the annealed gRNA molecules (500 nM) in cleavage assay buffer (20 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl₂, 1 mM DTT, 5% glycerol) in a total volume of 9 μl. Reactions are initiated by the addition of 1 μl target DNA (10 nM) and incubated for 1 h at 37° C. Reactions are quenched by the addition of 20 μl of loading dye (5 mM EDTA, 0.025% SDS, 5% glycerol in formamide) and heated to 95° C. for 5 min. Cleavage products are resolved on 12% denaturing polyacrylamide gels containing 7 M urea and visualized by phosphorimaging. The resulting cleavage products indicate that whether the complementary strand, the non-complementary strand, or both, are cleaved.

One or both of these assays can be used to evaluate the suitability of a candidate gRNA molecule or candidate Cas9 molecule.

Binding Assay: Testing the Binding of Cas9 Molecule to Target DNA

Exemplary methods for evaluating the binding of Cas9 molecule to target DNA are described, e.g., in Jinek et al. 2012 SCIENCE 337(6096): 816-821.

For example, in an electrophoretic mobility shift assay, target DNA duplexes are formed by mixing of each strand (10 nmol) in deionized water, heating to 95° C. for 3 min and slow cooling to room temperature. All DNAs are purified on 8% native gels containing 1×TBE. DNA bands are visualized by UV shadowing, excised, and eluted by soaking gel pieces in DEPC-treated H₂O. Eluted DNA is ethanol precipitated and dissolved in DEPC-treated H₂O. DNA samples are 5′ end labeled with [7-32P]-ATP using T4 polynucleotide kinase for 30 min at 37° C. Polynucleotide kinase is heat denatured at 65° C. for 20 min, and unincorporated radiolabel is removed using a column. Binding assays are performed in buffer containing 20 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl₂, 1 mM DTT and 10% glycerol in a total volume of 10 μl. Cas9 protein molecule is programmed with equimolar amounts of pre-annealed gRNA molecule and titrated from 100 μM to 1 μM. Radiolabeled DNA is added to a final concentration of 20 μM. Samples are incubated for 1 h at 37° C. and resolved at 4° C. on an 8% native polyacrylamide gel containing 1×TBE and 5 mM MgCl₂. Gels are dried and DNA visualized by phosphorimaging.

Repair Assays: Testing a Cas9 to Promote DNA Repair

The ability of a Cas9 system to promote DNA repair by a HDR pathway, e.g., HDR or ALT-HR, can be evaluated in a cell-based GFP assay. DNA repair by a HDR pathway is typically used to correct a gene with a mutation or undesired sequence. For this assay, a cell line carrying a non-functional GFP reporter system is used. An exogenous non-functional GFP gene, e.g., a GFP with an inactivating mutation, is delivered, e.g., by transfection, into a cell. Alternatively, the cell line carries one copy of a non-functional GFP gene integrated into the genome of the cell, e.g., by transduction. A candidate Cas9 protein molecule or nucleic acid, a gRNA that mediates binding of the Cas9 to the GFP gene to be corrected, and a template nucleic acid containing a functional, e.g., corrected GFP gene sequence, is delivered, e.g., by transfection, into the cell. An HDR-enhancer molecule is administered to the test cells, and no HDR-enhancer molecule is administered to control cells. The cells are cultured for a sufficient amount of time to allow repair and expression of the GFP gene, and GFP expression is analyzed by flow cytometry. An increase in GFP-expressing (GFP-positive) cells or an increased level of GFP signal, as compared to a control, indicates that DNA repair occurred, resulting in gene correction. GFP positive cells can be collected by cell sorting methods, and further analyzed by various sequencing methods to confirm correction of the targeted locus of the GFP gene.

The ability of a Cas9 to promote DNA repair by ALT-NHEJ can be evaluated in a cell-based GFP assay. DNA repair by the alt-NHEJ pathway is typically used to disrupt a gene and prevent expression. For this assay, a cell line carrying a functional GFP reporter system is used. An exogenous functional GFP gene, e.g., a wild-type GFP gene, is delivered, e.g., by transfection, into a cell. Alternatively, the cell line carries one copy of a functional or wild-type GFP gene integrated into the genome of the cell, e.g., by transduction. A candidate Cas9 protein molecule or nucleic acid and a gRNA that mediates binding of the Cas9 molecule to the GFP gene is delivered, e.g., by transfection, into the cell. An EPR-enhancer is administered to the test cells, and no EPR-enhancer is administered to control cells. The cells are cultured for a sufficient amount of time to allow repair and expression of the GFP gene, and GFP expression is analyzed by flow cytometry. A decrease in GFP-expressing cells or a decrease in the level of GFP signal, as compared to a control, indicates that DNA repair occurred, resulting in gene disruption. GFP negative cells can be collected by cell sorting methods, and further analyzed by various sequencing methods to confirm disruption of the targeted locus of the GFP gene.

Differential Scanning Flourimetry (DSF)

The thermostability of Cas9 molecule-gRNA ribonucleoprotein (RNP) complexes, e.g., a Cas9 molecule-gRNA RNP complex, can be measured via DSF. This technique measures the thermostability of a protein, which can increase under favorable conditions such as the addition of a binding molecule, e.g., a gRNA.

The assay is performed using two different protocols, one to test the best stoichiometric ratio of gRNA:Cas9 protein and another to determine the best solution conditions for RNP formation.

To determine the best solution to form RNP complexes, a 2 uM solution of Cas9 in water+10× SYPRO Orange® (Life Technologies cat #S-6650) and dispensed into a 384 well plate. An equimolar amount of gRNA diluted in solutions with varied pH and salt is then added. After incubating at room temperature for 10 min. and brief centrifugation to remove any bubbles, a Bio-Rad CFX384™ Real-Time System C1000 Touch™ Thermal Cycler with the Bio-Rad CFX Manager software is used to run a gradient from 20° C. to 90° C. with a 1° C. increase in temperature every 10 seconds.

The second assay consists of mixing various concentrations of gRNA with 2 uM Cas9 molecule in optimal buffer from the assay above and incubating at RT for 10 min in a 384 well plate. An equal volume of optimal buffer+10× SYPRO Orange® (Life Techonologies cat #S-6650) is added and the plate sealed with Microseal® B adhesive (MSB-1001). Following brief centrifugation to remove any bubbles, a Bio-Rad CFX384™ Real-Time System C1000 Touch™ Thermal Cycler with the Bio-Rad CFX Manager software is used to run a gradient from 20° C. to 90° C. with a 1° increase in temperature every 10 seconds.

Resection Assay: Testing a Cas9 to Promote Resection

The ability of a Cas9 to promote resection can be evaluated by measuring the levels of single stranded DNA at specific double strand break sites in human cells using quantitative methods (as described in Zhou et al., Nucleic Acids Res, 2014, 42(3):e19). In this assay, a cell line is delivered, e.g., by transfection, a candidate Cas9 or a candidate Cas9 fusion protein. The cells are cultured for a sufficient amount of time to allow nuclease activity and resection to occur. Genomic DNA is carefully extracted using a method in which cells are embedded in low-gelling point agar that protects the DNA from shearing and damage during extraction. The genomic DNA is digested with a restriction enzyme that selectively cuts double-stranded DNA. Primers for quantitative PCR that span up to 5 kb of the double strand break site are designed. The results from the PCR reaction show the levels of single strand DNA detected at each of the primer positions. Thus, the length and the level of resection promoted by the candidate Cas9 or Cas9 fusion protein can be determined from this assay.

Other qualitative assays for identifying the occurrence of resection include the detection of proteins or protein complexes that bind to single-stranded DNA after resection has occurred, e.g., RPA foci, Rad51 foci, or BrDU detection by immunofluorescence. Antibodies for RPA protein and Rad51 are known in the art.

V. Genome Editing Approaches

Mutations may be corrected, and undesirable nucleic acid sequences may be cleaved, using one of the approaches discussed herein. In an embodiment, a mutation in a target nucleic acid is corrected by homology directed repair (HDR) using a template nucleic acid (see Section V.1).

V.1 HDR Repair and Template Nucleic Acids

As described herein, nuclease-induced homology directed repair (HDR) can be used to alter a target sequence and correct (e.g., repair or edit) a mutation in the genome. While not wishing to be bound by theory, it is believed that alteration of the target sequence occurs by homology-directed repair (HDR) with a donor template or template nucleic acid. For example, the donor template or the template nucleic acid provides for alteration of the target position. It is contemplated that a plasmid donor can be used as a template for homologous recombination. In an embodiment where a double-stranded template nucleic acid is used, the target position is altered by HDR. It is further contemplated that a single stranded donor template can be used as a template for alteration of the target position by alternate methods of homology directed repair (e.g., HR, alt-HR, and/or single strand annealing) between the target position and the donor template. Donor template-effected alteration of a target position depends on target sequence cleavage by a Cas9 molecule. Cleavage by Cas9 can comprise a nick, a double strand break, or two single strand breaks, e.g., one on each strand of the target nucleic acid.

In an embodiment, a mutation can be corrected by either a single double-strand break or two single strand breaks. In an embodiment, a mutation can be corrected by (1) a single double-strand break, (2) two single strand breaks, (3) two double strand breaks with a break occurring on each side of the target position, (4) one double strand break and two single strand breaks with the double strand break and two single strand breaks occurring on each side of the target position (5) four single strand breaks with a pair of single strand breaks occurring on each side of the target position, or (6) one single strand break. In an embodiment where one single-stranded break is used, the target position can be altered by alternative HDR.

In an embodiment where a single-stranded template nucleic acid is used, the target position can be altered by alternative HDR.

Donor template-effected alteration of a target position depends on cleavage by a Cas9 molecule. Cleavage by Cas9 can comprise a nick, a double strand break, or two single strand breaks, e.g., one on each strand of the target nucleic acid. After introduction of the breaks on the target nucleic acid, resection occurs at the break ends resulting in single stranded overhanging DNA regions.

Methods of promoting HDR pathways, e.g., SSA, alt-HR, and/or HR, are described herein in Section VI.

Additional details on template nucleic acids are provided in Section IV entitled “Template nucleic acids” in International Application PCT/US2014/057905, Publication Number WO2015/048577.

Double Strand Break Mediated Correction

In an embodiment, double strand cleavage is effected by a Cas9 molecule having cleavage activity associated with an HNH-like domain and cleavage activity associated with a RuvC-like domain, e.g., an N-terminal RuvC-like domain, e.g., a wild type Cas9 molecule. Such embodiments require only a single gRNA. In other embodiments, dual gRNAs are required (see, for example, FIG. 2 ).

Single Strand Break Mediated Correction

In some embodiments, one single strand break, or nick, is effected by a Cas9 molecule having nickase activity, e.g., a Cas9 nickase as described herein. A nicked target nucleic acid can be a substrate for alt-HR.

In other embodiments, two single strand breaks, or nicks, are effected by a Cas9 molecule having nickase activity, e.g., cleavage activity associated with an HNH-like domain or cleavage activity associated with an N-terminal RuvC-like domain. Such embodiments usually require two gRNAs, one for placement of each single strand break. In an embodiment, the Cas9 molecule having nickase activity cleaves the strand to which the gRNA hybridizes, but not the strand that is complementary to the strand to which the gRNA hybridizes. In an embodiment, the Cas9 molecule having nickase activity does not cleave the strand to which the gRNA hybridizes, but rather cleaves the strand that is complementary to the strand to which the gRNA hybridizes.

In an embodiment, the nickase has HNH activity, e.g., a Cas9 molecule having the RuvC activity inactivated, e.g., a Cas9 molecule having a mutation at D10, e.g., the D10A mutation. D10A inactivates RuvC; therefore, the Cas9 nickase has (only) HNH activity and will cut on the strand to which the gRNA hybridizes (e.g., the complementary strand, which does not have the NGG PAM on it). In other embodiments, a Cas9 molecule having an H840, e.g., an H840A, mutation can be used as a nickase. H840A inactivates HNH; therefore, the Cas9 nickase has (only) RuvC activity and cuts on the non-complementary strand (e.g., the strand that has the NGG PAM and whose sequence is identical to the gRNA).

In other embodiments, a Cas9 molecule having an N863, e.g., the N863A mutation, mutation can be used as a nickase. N863A inactivates HNH therefore the Cas9 nickase has (only) RuvC activity and cuts on the non-complementary strand (the strand that has the NGG PAM and whose sequence is identical to the gRNA). In other embodiments, a Cas9 molecule having an N580, e.g., the N580A mutation, mutation can be used as a nickase. N580A inactivates HNH therefore the Cas9 nickase has (only) RuvC activity and cuts on the non-complementary strand (the strand that has the NGG PAM and whose sequence is identical to the gRNA). In an embodiment, in which a nickase and two gRNAs are used to position two single strand nicks, one nick is on the + strand and one nick is on the—strand of the target nucleic acid. The PAMs can be outwardly facing. The gRNAs can be selected such that the gRNAs are separated by, from about 0-50, 0-100, or 0-200 nucleotides. In an embodiment, there is no overlap between the target sequences that are complementary to the targeting domains of the two gRNAs. In an embodiment, the gRNAs do not overlap and are separated by as much as 50, 100, or 200 nucleotides. In an embodiment, the use of two gRNAs can increase specificity, e.g., by decreasing off-target binding (Ran et al. (2013) CELL 154: 1380-1389).

In an embodiment, a single nick can be used to induce HDR, e.g., alt-HR. It is contemplated herein that a single nick can be used to increase the ratio of HR to NHEJ at a given cleavage site. In an embodiment, a single strand break is formed in the strand of the target nucleic acid to which the targeting domain of said gRNA is complementary. In another embodiment, a single strand break is formed in the strand of the target nucleic acid other than the strand to which the targeting domain of said gRNA is complementary.

Placement of the Double Strand Break or a Single Strand Break Relative to Target Position or Other Landmark

The double strand break or single strand break in one of the strands should be sufficiently close to target position such that an alteration is produced in the desired region, e.g., correction of a mutation occurs. In an embodiment, the distance is not more than 50, 100, 200, 300, 350 or 400 nucleotides. While not wishing to be bound by theory, in some embodiments, it is believed that the break should be sufficiently close to target position such that the target position is within the region that is subject to exonuclease-mediated removal during end resection. If the distance between the target position and a break is too great, the mutation or other sequence desired to be altered may not be included in the end resection and, therefore, may not be corrected, as donor sequence, either exogenously provided donor sequence or endogenous genomic donor sequence, in some embodiments is only used to correct sequence within the end resection region.

In an embodiment, the targeting domain is configured such that a cleavage event, e.g., a double strand or single strand break, is positioned within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150 or 200 nucleotides of the region desired to be altered, e.g., a mutation. The break, e.g., a double strand or single strand break, can be positioned upstream or downstream of the region desired to be altered, e.g., a mutation. In some embodiments, a break is positioned within the region desired to be altered, e.g., within a region defined by at least two mutant nucleotides. In some embodiments, a break is positioned immediately adjacent to the region desired to be altered, e.g., immediately upstream or downstream of a mutation.

In an embodiment, a single strand break is accompanied by an additional single strand break, positioned by a second gRNA molecule, as discussed below. For example, the targeting domains bind configured such that a cleavage event, e.g., the two single strand breaks, are positioned within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150 or 200 nucleotides of a target position. In an embodiment, the first and second gRNA molecules are configured such, that when guiding a Cas9 nickase, a single strand break will be accompanied by an additional single strand break, positioned by a second gRNA, sufficiently close to one another to result in alteration of the desired region. In an embodiment, the first and second gRNA molecules are configured such that a single strand break positioned by said second gRNA is within 10, 20, 30, 40, or 50 nucleotides of the break positioned by said first gRNA molecule, e.g., when the Cas9 is a nickase. In an embodiment, the two gRNA molecules are configured to position cuts at the same position, or within a few nucleotides of one another, on different strands, e.g., essentially mimicking a double strand break.

In an embodiment, in which a gRNA (unimolecular (or chimeric) or modular gRNA) and Cas9 nuclease induce a double strand break for the purpose of inducing HDR-mediated correction, the cleavage site is between 0-200 bp (e.g., 0-175, 0 to 150, 0 to 125, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 25 to 200, 25 to 175, 25 to 150, 25 to 125, 25 to 100, 25 to 75, 25 to 50, 50 to 200, 50 to 175, 50 to 150, 50 to 125, 50 to 100, 50 to 75, 75 to 200, 75 to 175, 75 to 150, 75 to 125, 75 to 100 bp) away from the target position. In an embodiment, the cleavage site is between 0-100 bp (e.g., 0 to 75, 0 to 50, 0 to 25, 25 to 100, 25 to 75, 25 to 50, 50 to 100, 50 to 75 or 75 to 100 bp) away from the target position.

In some embodiments, one can promote HDR by using nickases to generate a break with overhangs. While not wishing to be bound by theory, the single stranded nature of the overhangs can enhance the cell's likelihood of repairing the break by HDR as opposed to, e.g., alt-NHEJ or c-NHEJ. Specifically, in some embodiments, HDR is promoted by selecting a first gRNA that targets a first nickase to a first target sequence, and a second gRNA that targets a second nickase to a second target sequence which is on the opposite DNA strand from the first target sequence and offset from the first nick.

In an embodiment, the targeting domain of a gRNA molecule is configured to position a cleavage event sufficiently far from a preselected nucleotide, e.g., the nucleotide of a coding region, such that the nucleotide is not altered. In an embodiment, the targeting domain of a gRNA molecule is configured to position an intronic cleavage event sufficiently far from an intron/exon border, or naturally occurring splice signal, to avoid alteration of the exonic sequence or unwanted splicing events. The gRNA molecule may be a first, second, third and/or fourth gRNA molecule, as described herein.

Placement of a First Break and a Second Break Relative to Each Other

In an embodiment, a double strand break can be accompanied by an additional double strand break, positioned by a second gRNA molecule, as is discussed below.

In an embodiment, a double strand break can be accompanied by two additional single strand breaks, positioned by a second gRNA molecule and a third gRNA molecule.

In an embodiment, a first and second single strand breaks can be accompanied by two additional single strand breaks positioned by a third gRNA molecule and a fourth gRNA molecule.

When two or more gRNAs are used to position two or more cleavage events, e.g., double strand or single strand breaks, in a target nucleic acid, it is contemplated that the two or more cleavage events may be made by the same or different Cas9 proteins. For example, when two gRNAs are used to position two double strand breaks, a single Cas9 nuclease may be used to create both double strand breaks. When two or more gRNAs are used to position two or more single strand breaks (nicks), a single Cas9 nickase may be used to create the two or more nicks. When two or more gRNAs are used to position at least one double strand break and at least one single strand break, two Cas9 proteins may be used, e.g., one Cas9 nuclease and one Cas9 nickase. It is contemplated that when two or more Cas9 proteins are used that the two or more Cas9 proteins may be delivered sequentially to control specificity of a double stranded versus a single strand break at the desired position in the target nucleic acid.

In some embodiments, the targeting domain of the first gRNA molecule and the targeting domain of the second gRNA molecules are complementary to opposite strands of the target nucleic acid molecule. In some embodiments, the gRNA molecule and the second gRNA molecule are configured such that the PAMs are oriented outward.

In certain embodiments, two gRNA are selected to direct Cas9-mediated cleavage at two positions that are a preselected distance from each other. In embodiments, the two points of cleavage are on opposite strands of the target nucleic acid. In some embodiments, the two cleavage points form a blunt ended break, and in other embodiments, they are offset so that the DNA ends comprise one or two overhangs (e.g., one or more 5′ overhangs and/or one or more 3′ overhangs). In some embodiments, each cleavage event is a nick. In embodiments, the nicks are close enough together that they form a break that is recognized by the double strand break machinery (as opposed to being recognized by, e.g., the SSBr machinery). In some embodiments, the nicks are far enough apart that they create an overhang that is a substrate for HDR, i.e., the placement of the breaks mimics a DNA substrate that has experienced some resection. For instance, in some embodiments the nicks are spaced to create an overhang that is a substrate for processive resection. In some embodiments, the two breaks are spaced within 25-65 nucleotides of each other. The two breaks may be, e.g., about 25, 30, 35, 40, 45, 50, 55, 60 or 65 nucleotides of each other. The two breaks may be, e.g., at least about 25, 30, 35, 40, 45, 50, 55, 60 or 65 nucleotides of each other. The two breaks may be, e.g., at most about 30, 35, 40, 45, 50, 55, 60 or 65 nucleotides of each other. In some embodiments, the two breaks are about 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, or 60-65 nucleotides of each other.

In some embodiments, the break that mimics a resected break comprises a 3′ overhang (e.g., generated by a DSB and a nick, where the nick leaves a 3′ overhang), a 5′ overhang (e.g., generated by a DSB and a nick, where the nick leaves a 5′ overhang), a 3′ and a 5′ overhang (e.g., generated by three cuts), two 3′ overhangs (e.g., generated by two nicks that are offset from each other), or two 5′ overhangs (e.g., generated by two nicks that are offset from each other).

In an embodiment, in which two gRNAs (independently, unimolecular (or chimeric) or modular gRNA) complexing with Cas9 nickases induce two single strand breaks for the purpose of inducing HDR-mediated correction, the closer nick is between 0-200 bp (e.g., 0-175, 0 to 150, 0 to 125, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 25 to 200, 25 to 175, 25 to 150, 25 to 125, 25 to 100, 25 to 75, 25 to 50, 50 to 200, 50 to 175, 50 to 150, 50 to 125, 50 to 100, 50 to 75, 75 to 200, 75 to 175, 75 to 150, 75 to 125, 75 to 100 bp) away from the target position and the two nicks will ideally be within 25-65 bp of each other (e.g., 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 30 to 55, 30 to 50, 30 to 45, 30 to 40, 30 to 35, 35 to 55, 35 to 50, 35 to 45, 35 to 40, 40 to 55, 40 to 50, 40 to 45 bp, 45 to 50 bp, 50 to 55 bp, 55 to 60 bp, 60 to 65 bp) and no more than 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20, 10 or 5 bp away from each other). In an embodiment, the cleavage site is between 0-100 bp (e.g., 0 to 75, 0 to 50, 0 to 25, 25 to 100, 25 to 75, 25 to 50, 50 to 100, 50 to 75 or 75 to 100 bp) away from the target position.

In one embodiment, two gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double-strand break on both sides of a target position. In an alternate embodiment, three gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double strand break (i.e., one gRNA complexes with a Cas9 nuclease) and two single strand breaks or paired single strand breaks (i.e., two gRNAs complex with Cas9 nickases) on either side of the target position. In another embodiment, four gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to generate two pairs of single strand breaks (i.e., two pairs of two gRNAs complex with Cas9 nickases) on either side of the target position. The double strand break(s) or the closer of the two single strand nicks in a pair will ideally be within 0-500 bp of the target position (e.g., no more than 450, 400, 350, 300, 250, 200, 150, 100, 50 or 25 bp from the target position). When nickases are used, the two nicks in a pair are, in embodiments, within 25-65 bp of each other (e.g., between 25 to 55, 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 35 to 45, 40 to 45 bp, 45 to 50 bp, 50 to 55 bp, 55 to 60 bp, or 60 to 65 bp) and no more than 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20 or 10 bp).

When two gRNAs are used to target Cas9 molecules to target DNA breaks, different combinations of Cas9 molecules are envisioned. In some embodiments, a first gRNA is used to target a first Cas9 molecule to a first target position, and a second gRNA is used to target a second Cas9 molecule to a second target position. In some embodiments, the first Cas9 molecule creates a nick on the first strand of the target nucleic acid, and the second Cas9 molecule creates a nick on the opposite strand, resulting in a double strand break (e.g., a blunt ended cut or a cut with overhangs).

Different combinations of nickases can be chosen to target one single strand break to one strand and a second single strand break to the opposite strand. When choosing a combination, one can take into account that there are nickases having one active RuvC-like domain, and nickases having one active HNH domain. In an embodiment, a RuvC-like domain cleaves the non-complementary strand of the target nucleic acid molecule. In an embodiment, an HNH-like domain cleaves a single stranded complementary domain, e.g., a complementary strand of a double stranded nucleic acid molecule. Generally, if both Cas9 molecules have the same active domain (e.g., both have an active RuvC domain or both have an active HNH domain), one will choose two gRNAs that bind to opposite strands of the target. In more detail, in some embodiments, a first gRNA is complementary with a first strand of the target nucleic acid and binds a nickase having an active RuvC-like domain and causes that nickase to cleave the strand that is non-complementary to that first gRNA, i.e., a second strand of the target nucleic acid; and a second gRNA is complementary with a second strand of the target nucleic acid and binds a nickase having an active RuvC-like domain and causes that nickase to cleave the strand that is non-complementary to that second gRNA, i.e., the first strand of the target nucleic acid. Conversely, in some embodiments, a first gRNA is complementary with a first strand of the target nucleic acid and binds a nickase having an active HNH domain and causes that nickase to cleave the strand that is complementary to that first gRNA, i.e., a first strand of the target nucleic acid; and a second gRNA is complementary with a second strand of the target nucleic acid and binds a nickase having an active HNH domain and causes that nickase to cleave the strand that is complementary to that second gRNA, i.e., the second strand of the target nucleic acid. In another arrangement, if one Cas9 molecule has an active RuvC-like domain and the other Cas9 molecule has an active HNH domain, the gRNAs for both Cas9 molecules can be complementary to the same strand of the target nucleic acid, so that the Cas9 molecule with the active RuvC-like domain will cleave the non-complementary strand and the Cas9 molecule with the HNH domain will cleave the complementary strand, resulting in a double strand break.

Exemplary Template Nucleic Acids

A template nucleic acid, as that term is used herein, refers to a nucleic acid sequence which can be used in conjunction with a Cas9 molecule and a gRNA molecule to alter the structure of a target position.

Target position, as used herein, refers to a site on a target nucleic acid (e.g., the chromosome) that is modified by a Cas9 molecule-dependent process. For example, the target position can be a modified Cas9 molecule cleavage of the target nucleic acid and template nucleic acid directed modification, e.g., correction, of the target position. In an embodiment, a target position can be a site between two nucleotides, e.g., adjacent nucleotides, on the target nucleic acid into which one or more nucleotides are added. The target position may comprise one or more nucleotides that are altered, e.g., corrected, by a template nucleic acid. In an embodiment, the target position is within a target sequence (e.g., the sequence to which the gRNA binds). In an embodiment, a target position is upstream or downstream of a target sequence (e.g., the sequence to which the gRNA binds).

In an embodiment, the target nucleic acid is modified to have the some or all of the sequence of the template nucleic acid, typically at or near cleavage site(s). In an embodiment, the template nucleic acid is single stranded. In an alternate embodiment, the template nucleic acid is double stranded. In an embodiment, the template nucleic acid is DNA, e.g., double stranded DNA. In an alternate embodiment, the template nucleic acid is single stranded DNA. In an embodiment, the template nucleic acid is encoded on the same vector backbone, e.g., AAV genome or plasmid DNA, as the Cas9 molecule and gRNA. In an embodiment, the template nucleic acid is excised from a vector backbone in vivo, e.g., it is flanked by gRNA recognition sequences. In an embodiment, the template nucleic acid comprises endogenous genomic sequence.

In an embodiment, the template nucleic acid alters the structure of the target position by participating in a homology directed repair event. In an embodiment, the template nucleic acid alters the sequence of the target position. In an embodiment, the template nucleic acid results in the incorporation of a modified, or non-naturally occurring base into the target nucleic acid.

Typically, the template sequence undergoes a breakage mediated or catalyzed recombination with the target sequence. In an embodiment, the template nucleic acid includes sequence that corresponds to a site on the target sequence that is cleaved by an eaCas9 mediated cleavage event. In an embodiment, the template nucleic acid includes sequence that corresponds to both, a first site on the target sequence that is cleaved in a first Cas9 mediated event, and a second site on the target sequence that is cleaved in a second Cas9 mediated event.

In an embodiment, the template nucleic acid can include sequence which results in an alteration in the coding sequence of a translated sequence, e.g., one which results in the substitution of one amino acid for another in a protein product, e.g., transforming a mutant allele into a wild type allele, transforming a wild type allele into a mutant allele, and/or introducing a stop codon, insertion of an amino acid residue, deletion of an amino acid residue, or a nonsense mutation.

In other embodiments, the template nucleic acid can include sequence which results in an alteration in a non-coding sequence, e.g., an alteration in an intron or in a 5′ or 3′ non-translated or non-transcribed region. Such alterations include an alteration in a control element, e.g., a promoter, enhancer, and an alteration in a cis-acting or trans-acting control element.

A template nucleic acid having homology with a target position in a gene, e.g., a gene described herein, can be used to alter the structure of a target sequence. The template sequence can be used to alter an unwanted structure, e.g., an unwanted or mutant nucleotide.

In an embodiment, the template nucleic acid is a single stranded nucleic acid. In another embodiment, the template nucleic acid is a double stranded nucleic acid. In some embodiments, the template nucleic acid comprises a nucleotide sequence, e.g., of one or more nucleotides, that will be added to or will template a change in the target nucleic acid. In other embodiments, the template nucleic acid comprises a nucleotide sequence that may be used to modify the target position. In other embodiments, the template nucleic acid comprises a nucleotide sequence, e.g., of one or more nucleotides, that corresponds to wild type sequence of the target nucleic acid, e.g., of the target position.

The template nucleic acid may comprise a replacement sequence. In some embodiments, the template nucleic acid comprises a 5′ homology arm. In other embodiments, the template nucleic acid comprises a 3′ homology arm.

In some embodiments, the template nucleic acid is linear double stranded DNA. The length may be, e.g., about 50-500 base pairs, e.g., about 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, or 500 base pairs. The length may be, e.g., at least 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, or 500 base pairs. In some embodiments, the length is no greater than 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, or 500 base pairs. In some embodiments, a double stranded template nucleic acid has a length of about 160 base pairs, e.g., about 155-165, 150-170, 140-180, 130-190, 120-200, 110-210, 100-220, 90-230, or 80-240 base pairs.

The template nucleic acid can be linear single stranded DNA. In some embodiments, the template nucleic acid is (i) linear single stranded DNA that can anneal to the nicked strand of the target nucleic acid, (ii) linear single stranded DNA that can anneal to the intact strand of the target nucleic acid, (iii) linear single stranded DNA that can anneal to the transcribed strand of the target nucleic acid, (iv) linear single stranded DNA that can anneal to the non-transcribed strand of the target nucleic acid, or more than one of the preceding. The length may be, e.g., 50-500 nucleotides. The length may be, e.g., about 50-500 nucleotides. The length may be, e.g., about 150-200 nucleotides, e.g., about 150, 160, 170, 180, 190, or 200 nucleotides. The length may be, e.g., at least 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, or 500 nucleotides. In some embodiments, the length is no greater than 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, or 500 nucleotides. In some embodiments, a single stranded template nucleic acid has a length of about 160 nucleotides, e.g., about 155-165, 150-170, 140-180, 130-190, 120-200, 110-210, 100-220, 90-230, or 80-240 nucleotides.

In some embodiments, the template nucleic acid is circular double stranded DNA, e.g., a plasmid. In some embodiments, the template nucleic acid comprises about 500 to 1000 base pairs of homology on either side of the replacement sequence and/or the nick. In some embodiments, the template nucleic acid comprises about 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology 5′ of the nick or replacement sequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick or replacement sequence. In some embodiments, the template nucleic acid comprises at least 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology 5′ of the nick or replacement sequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick or replacement sequence. In some embodiments, the template nucleic acid comprises no more than 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology 5′ of the nick or replacement sequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick or replacement sequence.

In some embodiments, the template nucleic acid is an adenovirus vector, e.g., an AAV vector, e.g., a ssDNA molecule of a length and sequence that allows it to be packaged in an AAV capsid. The vector may be, e.g., less than 5 kb and may contain an ITR sequence that promotes packaging into the capsid. The vector may be integration-deficient. In some embodiments, the template nucleic acid comprises about 150 to 1000 nucleotides of homology on either side of the replacement sequence and/or the nick. In some embodiments, the template nucleic acid comprises about 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5′ of the nick or replacement sequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick or replacement sequence. In some embodiments, the template nucleic acid comprises at least 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5′ of the nick or replacement sequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick or replacement sequence. In some embodiments, the template nucleic acid comprises at most 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5′ of the nick or replacement sequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick or replacement sequence.

In some embodiments, the template nucleic acid is a lentiviral vector, e.g., an IDLV (integration deficiency lentivirus). In some embodiments, the template nucleic acid comprises about 500 to 1000 base pairs of homology on either side of the replacement sequence and/or the nick. In some embodiments, the template nucleic acid comprises about 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology 5′ of the nick or replacement sequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick or replacement sequence. In some embodiments, the template nucleic acid comprises at least 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology 5′ of the nick or replacement sequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick or replacement sequence. In some embodiments, the template nucleic acid comprises no more than 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology 5′ of the nick or replacement sequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick or replacement sequence.

In some embodiments, the template nucleic acid comprises one or more mutations, e.g., silent mutations, that prevent Cas9 molecule from recognizing and cleaving the template nucleic acid. The template nucleic acid may comprise, e.g., at least 1, 2, 3, 4, 5, 10, 20, or 30 silent mutations relative to the corresponding sequence in the genome of the cell to be altered. In some embodiments, the template nucleic acid comprises at most 2, 3, 4, 5, 10, 20, 30, or 50 silent mutations relative to the corresponding sequence in the genome of the cell to be altered.

In an embodiment, the template nucleic acid alters the structure of the target position by participating in a homology directed repair event. In an embodiment, the template nucleic acid alters the sequence of the target position. In an embodiment, the template nucleic acid results in the incorporation of a modified, or non-naturally occurring base into the target nucleic acid.

Typically, the template sequence undergoes a breakage mediated or catalyzed recombination with the target sequence. In an embodiment, the template nucleic acid includes sequence that corresponds to a site on the target sequence that is cleaved by an eaCas9 mediated cleavage event. In an embodiment, the template nucleic acid includes sequence that corresponds to both, a first site on the target sequence that is cleaved in a first Cas9 mediated event, and a second site on the target sequence that is cleaved in a second Cas9 mediated event.

In an embodiment, the template nucleic acid can include sequence which results in an alteration in the coding sequence of a translated sequence, e.g., one which results in the substitution of one amino acid for another in a protein product, e.g., transforming a mutant allele into a wild type allele, transforming a wild type allele into a mutant allele, and/or introducing a stop codon, insertion of an amino acid residue, deletion of an amino acid residue, or a nonsense mutation. In an embodiment, the template nucleic acid can include sequence which results in an alteration in a coding sequence, e.g., an alteration in an exon.

In other embodiments, the template nucleic acid can include sequence which results in an alteration in a non-coding sequence, e.g., an alteration in an intron or in a 5′ or 3′ non-translated or non-transcribed region. Such alterations include an alteration in a control element, e.g., a promoter, enhancer, and an alteration in a cis-acting or trans-acting control element.

A template nucleic acid having homology with a target position can be used to alter the structure of a target sequence. The template sequence can be used to alter an unwanted structure, e.g., an unwanted or mutant nucleotide.

Length of the Homology Arms

The homology arm should extend at least as far as the region in which end resection may occur, e.g., in order to allow the resected single stranded overhang to find a complementary region within the donor template. The overall length could be limited by parameters such as plasmid size or viral packaging limits. In an embodiment, a homology arm does not extend into repeated elements, e.g., ALU elements or LINE elements.

Exemplary homology arm lengths include at least 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides. In some embodiments, the homology arm length is 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides.

A template nucleic acid typically comprises the following components:

[5′ homology arm]-[replacement sequence]-[3′ homology arm].

The homology arms provide for recombination into the chromosome, thus replacing the undesired element, e.g., a mutation or signature, with the replacement sequence. In an embodiment, the homology arms flank the most distal cleavage sites.

In an embodiment, the 3′ end of the 5′ homology arm is the position next to the 5′ end of the replacement sequence. In an embodiment, the 5′ homology arm can extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000 nucleotides 5′ from the 5′ end of the replacement sequence.

In an embodiment, the 5′ end of the 3′ homology arm is the position next to the 3′ end of the replacement sequence. In an embodiment, the 3′ homology arm can extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000 nucleotides 3′ from the 3′ end of the replacement sequence.

In an embodiment, to correct a mutation, the homology arms, e.g., the 5′ and 3′ homology arms, may each comprise about 1000 base pairs (bp) of sequence flanking the most distal gRNAs (e.g., 1000 bp of sequence on either side of the mutation).

It is contemplated herein that one or both homology arms may be shortened to avoid including certain sequence repeat elements, e.g., Alu elements or LINE elements. For example, a 5′ homology arm may be shortened to avoid a sequence repeat element. In other embodiments, a 3′ homology arm may be shortened to avoid a sequence repeat element. In some embodiments, both the 5′ and the 3′ homology arms may be shortened to avoid including certain sequence repeat elements.

It is contemplated herein that template nucleic acids for correcting a mutation may be designed for use as a single-stranded oligonucleotide, e.g., a single-stranded oligodeoxynucleotide (ssODN). When using a ssODN, 5′ and 3′ homology arms may range up to about 200 base pairs (bp) in length, e.g., at least 25, 50, 75, 100, 125, 150, 175, or 200 bp in length. Longer homology arms are also contemplated for ssODNs as improvements in oligonucleotide synthesis continue to be made. In some embodiments, a longer homology arm is made by a method other than chemical synthesis, e.g., by denaturing a long double stranded nucleic acid and purifying one of the strands, e.g., by affinity for a strand-specific sequence anchored to a solid substrate.

While not wishing to be bound by theory, in some embodiments HDR proceeds more efficiently when the template nucleic acid has extended homology 5′ to the nick (i.e., in the 5′ direction of the nicked strand) (see FIGS. 3 and 4 ). Accordingly, in some embodiments, the template nucleic acid has a longer homology arm and a shorter homology arm, wherein the longer homology arm can anneal 5′ of the nick. In some embodiments, the arm that can anneal 5′ to the nick is at least 25, 50, 75, 100, 125, 150, 175, or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000 nucleotides from the nick or the 5′ or 3′ end of the replacement sequence. In some embodiments, the arm that can anneal 5′ to the nick is at least 10%, 20%, 30%, 40%, or 50% longer than the arm that can anneal 3′ to the nick. In some embodiments, the arm that can anneal 5′ to the nick is at least 2×, 3×, 4×, or 5× longer than the arm that can anneal 3′ to the nick. Depending on whether a ssDNA template can anneal to the intact strand or the nicked strand, the homology arm that anneals 5′ to the nick may be at the 5′ end of the ssDNA template or the 3′ end of the ssDNA template, respectively.

Similarly, in some embodiments, the template nucleic acid has a 5′ homology arm, a replacement sequence, and a 3′ homology arm, such that the template nucleic acid has extended homology to the 5′ of the nick. For example, the 5′ homology arm and 3′ homology arm may be substantially the same length, but the replacement sequence may extend farther 5′ of the nick than 3′ of the nick. In some embodiments, the replacement sequence extends at least 10%, 20%, 30%, 40%, 50%, 2×, 3×, 4×, or 5x further to the 5′ end of the nick than the 3′ end of the nick.

While not wishing to be bound by theory, in some embodiments alt-HR proceeds more efficiently when the template nucleic acid is centered on the nick. Accordingly, in some embodiments, the template nucleic acid has two homology arms that are essentially the same size. For instance, the first homology arm of a template nucleic acid may have a length that is within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the second homology arm of the template nucleic acid.

Similarly, in some embodiments, the template nucleic acid has a 5′ homology arm, a replacement sequence, and a 3′ homology arm, such that the template nucleic acid extends substantially the same distance on either side of the nick. For example, the homology arms may have different lengths, but the replacement sequence may be selected to compensate for this. For example, the replacement sequence may extend further 5′ from the nick than it does 3′ of the nick, but the homology arm 5′ of the nick is shorter than the homology arm 3′ of the nick, to compensate. The converse is also possible, e.g., that the replacement sequence may extend further 3′ from the nick than it does 5′ of the nick, but the homology arm 3′ of the nick is shorter than the homology arm 5′ of the nick, to compensate.

Exemplary Arrangements of Linear Nucleic Acid Template Systems

In an embodiment, the nucleic acid template system is double stranded. In an embodiment, the nucleic acid template system is single stranded. In an embodiment, the nucleic acid template system comprises a single stranded portion and a double stranded portion.

In an embodiment, the template nucleic acid comprises about 50 to 500 base pairs. In an embodiment, the template nucleic acid comprises about 50 to 100, e.g., 55 to 95, 60 to 90, 65 to 85, or 70 to 80, base pairs, homology on either side of the nick and/or replacement sequence. In an embodiment, the template nucleic acid comprises about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs homology 5′ of the nick or replacement sequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick or replacement sequences.

In an embodiment, the template nucleic acid comprises about 150 to 200, e.g., 155 to 195, 160 to 190, 165 to 185, or 170 to 180, base pairs homology 3′ of the nick and/or replacement sequence. In an embodiment, the template nucleic acid comprises about 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 base pairs homology 3′ of the nick or replacement sequence. In an embodiment, the template nucleic acid comprises less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, or 10 base pairs homology 5′ of the nick or replacement sequence.

In an embodiment, the template nucleic acid comprises about 150 to 200, e.g., 155 to 195, 160 to 190, 165 to 185, or 170 to 180, base pairs homology 5′ of the nick and/or replacement sequence. In an embodiment, the template nucleic acid comprises about 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 base pairs homology 5′ of the nick or replacement sequence. In an embodiment, the template nucleic acid comprises less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, or 10 base pairs homology 3′ of the nick or replacement sequence.

Exemplary Arrangements of Circular Nucleic Acid Template Systems

In an embodiment, the nucleic acid template system is double stranded. In an embodiment, the nucleic acid template system is double stranded comprises a single stranded portion and a double stranded portion. In an embodiment, the nucleic acid template system is single stranded. In one embodiment, the nucleic acid template system is a plasmid. In another embodiment, the nucleic acid template is an endogenous nucleic acid. In another embodiment, the nucleic acid template is present in an AAV or an IDLV.

In an embodiment, the template nucleic acid comprises about 500 to 2000, e.g., 700 to 1900, 800 to 1800, 900 to 1700, 900 to 1600, 1000 to 1500, 1100 to 1400, or 1200 to 1300 base pairs, homology on either side of the nick and/or replacement sequence. In an embodiment, the template nucleic acid comprises about 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 base pairs homology 5′ of the nick or replacement sequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick or replacement sequences.

In an embodiment, the template nucleic acid comprises about 500 to 2000, e.g., 700 to 1900, 800 to 1800, 900 to 1700, 900 to 1600, 1000 to 1500, 1100 to 1400, or 1200 to 1300, base pairs homology 3′ of the nick and/or replacement sequence. In an embodiment, the template nucleic acid comprises about 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 base pairs homology 3′ of the nick or replacement sequence. In an embodiment, the template nucleic acid comprises less than about 500, 400, 300, 200, 100, or 50 base pairs homology 5′ of the nick or replacement sequence.

In an embodiment, the template nucleic acid comprises about 500 to 2000, e.g., 700 to 1900, 800 to 1800, 900 to 1700, 900 to 1600, 1000 to 1500, 1100 to 1400, or 1200 to 1300, base pairs homology 5′ of the nick and/or replacement sequence. In an embodiment, the template nucleic acid comprises about 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 base pairs homology 5′ of the nick or replacement sequence. In an embodiment, the template nucleic acid comprises less than about 500, 400, 300, 200, 100, or 50 base pairs homology 3′ of the nick or replacement sequence.

Methods of Promoting Break Repair by an HDR Pathway

In another aspect, disclosed herein is a method of altering a cell, e.g., altering the structure, e.g., altering the sequence, of a target nucleic acid of a cell, comprising contacting the cell with: (a) a gRNA that targets a target position, e.g., a gRNA as described herein; (b) a Cas9 molecule, e.g., a Cas9 molecule as described herein; (c) a template nucleic acid, (d) an HDR-enhancer and optionally, (e) a second, third and/or fourth gRNA, as described herein. The methods can comprise contacting said cell with (a) and (b). The methods can comprise contacting said cell with (a), (b), and (c), and/or with (a), (b), (c) and (d).

The contacting may be performed ex vivo and the contacted cell may be returned to the subject's body after the contacting step. In other embodiments, the contacting step may be performed in vivo.

In some embodiments, contacting a cell with a Cas9 molecule comprises contacting the cell with a nucleic acid encoding the Cas9 molecule and allowing the cell to produce the Cas9 molecule. In some embodiments, contacting a cell with a gRNA comprises contacting the cell with DNA that can direct transcription of the gRNA, and allowing the cell to produce the gRNA.

In some embodiments, the method of altering a cell, as described herein, comprises acquiring knowledge of the presence of an undesired sequence in said cell, prior to the contacting step. Acquiring knowledge of the sequence of the undesired sequence in the cell may be by DNA sequencing.

In some embodiments, the contacting step of the method comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, that expresses or comprises at least one of (a), (b), (c), (d), and optionally (e). In some embodiments, the contacting step of the method comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, that expresses or comprises each of (a), (b), (c), (d), and optionally (e). In some embodiments, the contacting step of the method comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, that expresses or comprises two, three, or four of (a), (b), (c), (d), and optionally (e). In some embodiments, the contacting step of the method comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, that expresses or comprises each of (a) and (b). In another embodiment, the contacting step of the method comprises delivering to the cell a Cas9 molecule of (b), a nucleic acid which encodes a gRNA according to (a), and a template nucleic acid of (c), optionally a HDR-enhancer of (d), and optionally, a second gRNA (e)(i) (and further optionally, a third gRNA (e)(iv) and/or fourth gRNA (e)(iii)).

The contacting step of the method can comprise contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, that expresses or comprises at least one of (a), (b), (c) and (d). In some embodiments, the contacting step of the method comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, that expresses each of (a), (b), and (c). In another embodiment, the contacting step of the method comprises delivering to the cell a Cas9 molecule of (b), a nucleic acid which encodes a gRNA (a) and a template nucleic acid of (c), optionally an HDR-enhancer of (d), and optionally, a second gRNA (e)(i) (and further optionally, a third gRNA (e)(iv) and/or fourth gRNA (e)(iii).

In an embodiment, contacting comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, e.g., an AAV2 vector, a modified AAV2 vector, an AAV3 vector, a modified AAV3 vector, an AAV6 vector, a modified AAV6 vector, an AAV8 vector or an AAV9 vector.

In an embodiment, contacting comprises delivering to the cell a Cas9 molecule, as a protein or an mRNA, and a nucleic acid which encodes or comprises a gRNA molecule and an HDR-enhancer molecule, and optionally a template nucleic acid and/or a second gRNA molecule.

In some embodiments, contacting comprises delivering to the cell a Cas9 molecule of (b), as a protein or an mRNA, said gRNA of (a), as an RNA, and optionally said second gRNA of (e), as an RNA, and optionally (c) as a nucleic acid. In some embodiments, the HDR-enhancer of (d) is delivered as a nucleic acid, e.g., DNA or RNA. In some embodiments, the HDR-enhancer of (d) is delivered as a functional nucleic acid such as a siRNA or RNAi oligonucleotide. In some embodiments, the HDR-enhancer of (d) is delivered as a nucleic acid, e.g., mRNA, that encodes a protein. In some embodiments, the HDR-enhancer molecule is delivered as a HDR-enhancing gRNA. In some embodiments, the HDR-enhancing gRNA is delivered in combination with an eiCas9 molecule. In some embodiments, the HDR-enhancing gRNA is delivered in combination with an eaCas9 molecule.

In some embodiment, contacting comprises delivering to the cell a gRNA of (a) as an RNA, optionally said second gRNA of (e) as an RNA, and a nucleic acid that encodes the Cas9 molecule of (b), (c) as a nucleic acid, and optionally (d) as a nucleic acid.

In some embodiments, a subject is treated by inducing a Cas9-mediated break at a target position, wherein the target position causes or exacerbates a disease or disorder, and administering a template nucleic acid and an HDR enhancer, wherein the break is repaired by HDR.

The method of treating a subject may comprise contacting the subject (or a cell from the subject) with (c) a template nucleic acid. A template nucleic acid is used when the method of treating a subject uses HDR to alter the sequence of the target nucleic acid of the subject.

In an embodiment, the method comprises acquiring knowledge of an undesired sequence in said subject, e.g., by DNA sequencing.

In an embodiment, the method comprises correcting an undesired sequence by HDR.

When the method comprises correcting an undesired sequence by HDR, a Cas9 molecule of (b), at least one guide RNA, e.g., a guide RNA of (a) and a template nucleic acid (c) can be included in the contacting step.

In an embodiment, a cell of the subject is contacted ex vivo with one or more of, e.g., all of (a), (b), (c) and (d). In an embodiment, said cell is returned to the subject's body.

In an embodiment, a cell of the subject is contacted is in vivo with one or more of, e.g., all of (a), (b) (c) and (d).

In an embodiment, the cell of the subject can also be contacted in vivo by intravenous delivery of one or more of, e.g., all of (a), (b), (c), and (d).

In an embodiment, contacting comprises contacting the subject with a nucleic acid, e.g., a vector, e.g., an AAV vector, described herein, e.g., a nucleic acid that encodes or comprises at least one of, e.g., all of, (a), (b), (c), and (d).

In an embodiment, contacting comprises delivering to said subject said Cas9 molecule of (b), as a protein or mRNA, and one or more nucleic acid which encodes or comprises at least one of, e.g., all of, (a), (c), and (d).

In an embodiment, contacting comprises delivering to the subject the Cas9 molecule of (b), as a protein or mRNA, the gRNA of (a), as an RNA, a nucleic acid of (c) and optionally the HDR-enhancer of (d), as an RNA.

In an embodiment, contacting comprises delivering to the subject the gRNA of (a), as an RNA, a nucleic acid that encodes the Cas9 molecule of (b), a nucleic acid of (c), and optionally an HDR-enhancer of (d).

In an embodiment, a cell of the subject can be contacted ex vivo with (a), (b), (c) and (d). In an embodiment, said cell is returned to the subject's body.

In an embodiment, contacting comprises contacting the subject with a nucleic acid, e.g., a vector, e.g., an AAV vector, described herein, e.g., a nucleic acid that encodes or comprises at least one, e.g., all of, of (a), (b), (c), and (d).

In an embodiment, contacting comprises delivering to the subject the Cas9 molecule of (b), as a protein or mRNA, and a nucleic acid which encodes or comprises one or more of (a), (c), and (d).

In an embodiment, contacting comprises delivering to the subject the Cas9 molecule of (b), as a protein or mRNA, the gRNA of (a), as an RNA, and the template nucleic acid of (c) as a DNA.

In an embodiment, contacting comprises delivering to the subject the gRNA of (a), as an RNA, optionally said second gRNA of (e), as an RNA, and a nucleic acid that encodes the Cas9 molecule of (b), and a nucleic acid that encodes the template nucleic acid of (d).

Examples of gRNAs in Genome Editing Methods

gRNA molecules as described herein can be used with Cas9 molecules that generate a double strand break or a single strand break to alter the sequence of a target nucleic acid, e.g., a target position. gRNA molecules useful in these methods are described below.

In some embodiments, the gRNA is used in making double strand breaks. In an embodiment, the gRNA, e.g., a chimeric gRNA, is configured such that it comprises one or more of the following properties:

-   -   a) it can position, e.g., when targeting a Cas9 molecule that         makes double strand breaks, a double strand break (i) within 50,         100, 150 or 200 nucleotides of a target position, or (ii)         sufficiently close that the target position is within the region         of end resection;     -   b) it has a targeting domain of at least 17 nucleotides, e.g., a         targeting domain of (i) 17, (ii) 18, or (iii) 20 nucleotides;         and     -   c)     -   (i) the proximal and tail domain, when taken together, comprise         at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53         nucleotides, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45,         49, 50, or 53 nucleotides from a naturally occurring S.         aureus, S. thermophilus, or N. meningitidis tail and proximal         domain, or a sequence that differs by no more than 1, 2, 3, 4,         5, 6, 7, 8, 9 or 10 nucleotides therefrom;     -   (ii) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,         50, or 53 nucleotides 3′ to the last nucleotide of the second         complementarity domain, e.g., at least 15, 18, 20, 25, 30, 31,         35, 40, 45, 49, 50, or 53 nucleotides from the corresponding         sequence of a naturally occurring S. aureus, S. thermophilus,         or N. meningitidis gRNA, or a sequence that differs by no more         than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;     -   (iii) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,         51, or 54 nucleotides 3′ to the last nucleotide of the second         complementarity domain that is complementary to its         corresponding nucleotide of the first complementarity domain,         e.g., at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54         nucleotides from the corresponding sequence of a naturally         occurring S. aureus, S. thermophilus, or N. meningitidis gRNA,         or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7,         8, 9 or 10 nucleotides therefrom;     -   iv) the tail domain is at least 10, 15, 20, 25, 30, 35 or 40         nucleotides in length, e.g., it comprises at least 10, 15, 20,         25, 30, 35 or 40 nucleotides from a naturally occurring S.         aureus, S. thermophilus, or N. meningitidis tail domain; or a         sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9         or 10 nucleotides therefrom; or     -   (v) the tail domain comprises 15, 20, 25, 30, 35, 40 nucleotides         or all of the corresponding portions of a naturally occurring         tail domain, e.g., a naturally occurring S. aureus, S.         thermophilus, or N. meningitidis tail domain.

In an embodiment, the gRNA is configured such that it comprises properties: a and b(i).

In an embodiment, the gRNA is configured such that it comprises properties: a and b(ii).

In an embodiment, the gRNA is configured such that it comprises properties: a and b(iii).

In an embodiment, the gRNA is configured such that it comprises properties: a and c.

In an embodiment, the gRNA is configured such that in comprises properties: a, b, and c.

In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(i).

In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(ii).

In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(i).

In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(ii).

In some embodiments, the gRNA is used in making single strand breaks. In an embodiment, the gRNA, e.g., a chimeric gRNA, is configured such that it comprises one or more of the following properties:

-   -   a) it can position, e.g., when targeting a Cas9 molecule that         makes single strand breaks, a single strand break (i) within 50,         100, 150 or 200 nucleotides of a target position, or (ii)         sufficiently close that the target position is within the region         of end resection;     -   b) it has a targeting domain of at least 17 nucleotides, e.g., a         targeting domain of (i) 17, (ii) 18, or (iii) 20 nucleotides;         and     -   c)     -   (i) the proximal and tail domain, when taken together, comprise         at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53         nucleotides, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45,         49, 50, or 53 nucleotides from a naturally occurring S.         aureus, S. thermophilus, or N. meningitidis tail and proximal         domain, or a sequence that differs by no more than 1, 2, 3, 4,         5, 6, 7, 8, 9 or 10 nucleotides therefrom;     -   (ii) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,         50, or 53 nucleotides 3′ to the last nucleotide of the second         complementarity domain, e.g., at least 15, 18, 20, 25, 30, 31,         35, 40, 45, 49, 50, or 53 nucleotides from the corresponding         sequence of a naturally occurring S. aureus, S. thermophilus,         or N. meningitidis gRNA, or a sequence that differs by no more         than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;     -   (iii) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,         51, or 54 nucleotides 3′ to the last nucleotide of the second         complementarity domain that is complementary to its         corresponding nucleotide of the first complementarity domain,         e.g., at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54         nucleotides from the corresponding sequence of a naturally         occurring S. aureus, S. thermophilus, or N. meningitidis gRNA,         or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7,         8, 9 or 10 nucleotides therefrom;     -   iv) the tail domain is at least 10, 15, 20, 25, 30, 35 or 40         nucleotides in length, e.g., it comprises at least 10, 15, 20,         25, 30, 35 or 40 nucleotides from a naturally occurring S.         aureus, S. thermophilus, or N. meningitidis tail domain; or, a         sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9         or 10 nucleotides therefrom; or     -   (v) the tail domain comprises 15, 20, 25, 30, 35, 40 nucleotides         or all of the corresponding portions of a naturally occurring         tail domain, e.g., a naturally occurring S. aureus, S.         thermophilus, or N. meningitidis tail domain.

In an embodiment, the gRNA is configured such that it comprises properties: a and b(i).

In an embodiment, the gRNA is configured such that it comprises properties: a and b(ii).

In an embodiment, the gRNA is configured such that it comprises properties: a and b(iii).

In an embodiment, the gRNA is configured such that it comprises properties: a and c.

In an embodiment, the gRNA is configured such that in comprises properties: a, b, and c.

In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(i).

In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(ii).

In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(i).

In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(ii).

In an embodiment, the gRNA is used with a Cas9 nickase molecule having HNH activity, e.g., a Cas9 molecule having the RuvC activity inactivated, e.g., a Cas9 molecule having a mutation at D10, e.g., the D10A mutation.

In an embodiment, the gRNA is used with a Cas9 nickase molecule having RuvC activity, e.g., a Cas9 molecule having the HNH activity inactivated, e.g., a Cas9 molecule having a mutation at H840, e.g., a H840A.

In an embodiment, a pair of gRNAs, e.g., a pair of chimeric gRNAs, comprising a first and a second gRNA, is configured such that they comprises one or more of the following properties:

-   -   a) one or both of the gRNAs can position, e.g., when targeting a         Cas9 molecule that makes single strand breaks, a single strand         break within (i) 50, 100, 150 or 200 nucleotides of a target         position, or (ii) sufficiently close that the target position is         within the region of end resection;     -   b) one or both have a targeting domain of at least 17         nucleotides, e.g., a targeting domain of (i) 17 or (ii) 18         nucleotides;     -   c) for one or both:     -   (i) the proximal and tail domain, when taken together, comprise         at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53         nucleotides, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45,         49, 50, or 53 nucleotides from a naturally occurring S.         aureus, S. thermophilus, or N. meningitidis tail and proximal         domain, or a sequence that differs by no more than 1, 2, 3, 4,         5, 6, 7, 8, 9 or 10 nucleotides therefrom;     -   (ii) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,         50, or 53 nucleotides 3′ to the last nucleotide of the second         complementarity domain, e.g., at least 15, 18, 20, 25, 30, 31,         35, 40, 45, 49, 50, or 53 nucleotides from the corresponding         sequence of a naturally occurring S. aureus, S. thermophilus,         or N. meningitidis gRNA, or a sequence that differs by no more         than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;     -   (iii) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,         51, or 54 nucleotides 3′ to the last nucleotide of the second         complementarity domain that is complementary to its         corresponding nucleotide of the first complementarity domain,         e.g., at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54         nucleotides from the corresponding sequence of a naturally         occurring S. aureus, S. thermophilus, or N. meningitidis gRNA,         or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7,         8, 9 or 10 nucleotides therefrom;     -   iv) the tail domain is at least 10, 15, 20, 25, 30, 35 or 40         nucleotides in length, e.g., it comprises at least 10, 15, 20,         25, 30, 35 or 40 nucleotides from a naturally occurring S.         aureus, S. thermophilus, or N. meningitidis tail domain; or a         sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9         or 10 nucleotides therefrom; or     -   (v) the tail domain comprises 15, 20, 25, 30, 35, 40 nucleotides         or all of the corresponding portions of a naturally occurring         tail domain, e.g., a naturally occurring S. aureus, S.         thermophilus, or N. meningitidis tail domain;     -   d) the gRNAs are configured such that, when hybridized to target         nucleic acid, they are separated by 0-50, 0-100, 0-200, at least         10, at least 20, at least 30 or at least 50 nucleotides;     -   e) the breaks made by the first gRNA and second gRNA are on         different strands; and     -   f) the PAMs are facing outwards.

In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(i). In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(ii). In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(iii). In an embodiment, one or both of the gRNAs configured such that it comprises properties: a and c. In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a, b, and c. In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(i), and c(i). In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(i), and c(ii). In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(i), c, and d. In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(i), c, and e. In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(i), c, and f. In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(i), c, d, and e. In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(i), c, d, and f. In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(i), c, d, e, and f. In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(iii), and c(i). In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(iii), and c(ii). In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(iii), c, and d. In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(iii), c, and e. In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(iii), c, and f. In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(iii), c, d, and e. In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(iii), c, d, and f. In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(iii), c, d, e, and f.

In an embodiment, the gRNAs are used with a Cas9 nickase molecule having HNH activity, e.g., a Cas9 molecule having the RuvC activity inactivated, e.g., a Cas9 molecule having a mutation at D10, e.g., the D10A mutation.

In an embodiment, the gRNAs are used with a Cas9 nickase molecule having RuvC activity, e.g., a Cas9 molecule having the HNH activity inactivated, e.g., a Cas9 molecule having a mutation at H840, e.g., a H840A.

V.2 Non-Homologous End-Joining

Nuclease-induced non-homologous end-joining (NHEJ), for example alt-NHEJ or canonical NHEJ, can be used to target gene-specific knockouts (see FIG. 1 ). NHEJ, e.g., alt-NHEJ, can also be used to remove (e.g., delete) sequence in a gene of interest. In the methods for altering a cell or treating a subject by altering a cell described herein, the cell is contacted with a Cas9 molecule described herein in an amount and under conditions sufficient for NHEJ, e.g., alt-NHEJ, to occur. In an embodiment, a deletion is inserted into the nucleic acid of the cell, thereby altering the sequence of the nucleic acid of the cell. In one embodiment, Alt-NHEJ pathways include blunt EJ, MMEJ, and SD-MMEJ (see FIG. 1 ).

While not wishing to be bound by theory, it is believed that, in an embodiment, the genomic alterations associated with the methods described herein rely on nuclease-induced NHEJ, e.g., alt-NHEJ, and the error-prone nature of the alt-NHEJ repair pathway. NHEJ repairs a double-strand break in the DNA by joining together the two ends; however, generally, the original sequence is restored only if two compatible ends, exactly as they were formed by the double-strand break, are perfectly ligated. The DNA ends of the double-strand break are frequently the subject of enzymatic processing, resulting in the addition or removal of nucleotides, e.g., resection, at one or both strands, prior to rejoining of the ends. This results in the presence of insertion and/or deletion (indel) mutations in the DNA sequence at the site of the NHEJ repair. Two-thirds of these mutations typically alter the reading frame and, therefore, produce a non-functional protein. Additionally, mutations that maintain the reading frame, but which insert or delete a significant amount of sequence, can destroy functionality of the protein. This is locus dependent as mutations in critical functional domains are likely less tolerable than mutations in non-critical regions of the protein.

The indel mutations generated by NHEJ are unpredictable in nature; however, at a given break site certain indel sequences are favored and are over represented in the population, likely due to small regions of microhomology. The lengths of deletions can vary widely; most commonly in the 1-50 bp range, but they can easily reach greater than 100-200 bp. Insertions tend to be shorter and often include short duplications of the sequence immediately surrounding the break site. However, it is possible to obtain large insertions, and in these cases, the inserted sequence has often been traced to other regions of the genome or to plasmid DNA present in the cells.

Because NHEJ, e.g., alt-NHEJ, is a mutagenic process, it can also be used to delete small sequence motifs as long as the generation of a specific final sequence is not required. If a double-strand break is targeted near to a short target sequence, the deletion mutations caused by the NHEJ repair often span, and therefore remove, the unwanted nucleotides. For the deletion of larger DNA segments, introducing two double-strand breaks, one on each side of the sequence, can result in NHEJ between the ends with removal of the entire intervening sequence. Both of these approaches can be used to delete specific DNA sequences; however, the error-prone nature of NHEJ may still produce indel mutations at the site of repair.

Two distinct NHEJ pathways are described herein, canonical NHEJ and alternative NHEJ. Canonical NHEJ typically occurs when a double strand break has blunt, unresected ends that are ligation-competent. In some instances, minimal end processing, e.g., <5 nucleotide deletions or insertions, occurs, and the break ends are ligated thereby resulting in either correct (error-free) repair, or approximately 1-4 nucleotide insertions or deletions. Canonical NHEJ is dependent upon the KU70/80 and XRCC4/LigaseIV pathway for recognition of the break, minimal end processing, DNA synthesis, and ligation.

In contrast, alternative NHEJ is not dependent upon the KU70/80 and XRCC4/LigaseIV pathway and typically occurs when resection of more than 5 nucleotides at the break ends occurs. In some cases, resection reveals a short span, e.g., 5 to 25 nucleotides, of homologous sequence in the overhangs, also known as microhomologies. The microhomologies anneal and the intervening sequence on the single strands between the break and the annealed microhomology region is deleted. Accordingly, ALT-NHEJ typically results in longer stretches, e.g., greater than 5 nucleotides, of deleted sequence than canonical NHEJ.

Both double strand cleaving eaCas9 molecules and single strand, or nickase, eaCas9 molecules can be used in the methods and compositions described herein to generate NHEJ-mediated indels. NHEJ-mediated indels targeted to a gene, e.g., a coding region, e.g., an early coding region of a gene of interest can be used to knockout (i.e., eliminate protein expression of) a gene of interest. For example, early coding region of a gene of interest includes sequence immediately following a transcription start site, within a first exon of the coding sequence, or within 500 bp of the transcription start site (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp).

V.3 Single-Strand Annealing

Single strand annealing (SSA) is a type of HDR-repair. Specifically, SSA repairs a double-strand break between two repeat sequences present in a target nucleic acid. Repeat sequences utilized by the SSA pathway are generally greater than 30 nucleotides in length. Resection at the break ends occurs to reveal repeat sequences on both strands of the target nucleic acid. After resection, single strand overhangs containing the repeat sequences are coated with RPA protein to prevent the repeats sequences from inappropriate annealing, e.g., to themselves. RAD52 binds to each of the repeat sequences on the overhangs and aligns the sequences to enable the annealing of the complementary repeat sequences. After annealing, the single-strand flaps of the overhangs are cleaved. New DNA synthesis fills in any gaps, and ligation restores the DNA duplex. As a result of the processing, the DNA sequence between the two repeats is deleted. The length of the deletion can depend on many factors including the location of the two repeats utilized, and the pathway or processivity of the resection.

SSA does not require a template nucleic acid to alter or correct a target nucleic acid sequence. Instead, the complementary repeat sequence is utilized.

VI. Methods of Promoting an HDR Repair Pathway

This section describes the machinery involved in different DNA repair pathways, and ways of modulating that machinery. While not wishing the theory in this section to be binding, it is believed that in many cases, inhibition of the steps or factors involved in a first DNA repair pathway increases the likelihood that a lesion will be repaired by another DNA repair pathway (e.g., HDR, such as HR, alt-HR, and/or SSA). Additional details on the DNA repair machinery are found in Ciccia and Elledge (2010) MOL. CELL 40(2): 179-204.

To begin, the relationships between different DNA damage repair pathways are described.

When a cell encounters a DSB, it follows a two-step model. The first step is the choice between canonical NHEJ (sometimes abbreviated herein as C-NHEJ) which operates on blunt DNA ends (i.e., no resection), and the initiation of resection of the DNA at the site of the DSB. After the resection is engaged, the cell faces a choice between alternative non-homologous end-joining (Alt-NHEJ, which is a class of repair pathways that includes MMEJ) and HDR (which is a class of repair pathways that includes HR, alt-HR and SSA). KU70-80 is a protein complex that has affinity for double-strand breaks and it is one of the key factors that regulates canonical NHEJ, and it suppresses both HR and alt-NHEJ. The length of resection of the DNA and the state of the cell cycle have an important role in engaging HDR versus Alt-NHEJ. Specifically, longer resection is required for HDR (hundreds of nucleotides) whereas typically short resection is needed for Alt-NHEJ; HR is active in S and G2 instead Alt-NHEJ is active throughout the cell cycle.

Each pathway is described in more detail in the following sections and tables.

TABLE VI.1 Representative Targets in Damage Repair Pathways Compound/siRNA/ Inhibition of Will promote Target gRNA/antiMiR (A) Inhibition of components Increase Resection: 53 BP1 siRNA/gRNA of the DNA damage Increase of ALT- Dominant negative response that prevents NHEJ, HDR. Balance peptide resection at the Break between ALT-NHEJ RIf1 siRNA/gRNA promoting indirectly and HDR will depend PTIP siRNA/gRNA NHEJInhibition of if there is homology components of the DNA (either the sister damage response that chromatin or the prevents resection at the donor) Break promoting indirectly NHEJ + B3:C51 (B) Inhibition of Canonical Increase Resection: KU 70-80 siRNA/gRNA NHEJ Increase of ALT- DNApk NU7441 NHEJ, HDR. Balance CC115 between ALT-NHEJ NK314 and HDR will depend Wortmannin if there is homology LY294002 (either the sister NU 7026 chromatin or the IC86621 donor) IC87102 IC87361 OK1035 SU11752 IC486241 Vaillin Lig4 SCR7 XRCC4 siRNA/gRNA XLF siRNA/gRNA Artemis siRNA/gRNA (C) Inhibition of HR Increase cNHEJ, ALT- BRCA2 siRNA/gRNA NHEJ/SSA and ALT- BRCA1 siRNA/gRNA HR CtIP siRNA/gRNA dominant negative protein EXo1 siRNA/gRNA DNA2 siRNA/gRNA MRN siRNA/gRNA complex MRE11 Mirin Telomelysin Resveratrol siRNA/gRNA Rad50 siRNA/gRNA Nbs1 siRNA/gRNA Rad51 B02 A03 RI-1 IBR2 siRNA/gRNA (J) ALT-NHEJ Increase of HDR: ALT- XRCC1 siRNA/gRNA HR, SSA, HR Ligase I siRNA/gRNA Ligase III siRNA/gRNA Pol Theta siRNA/gRNA (D) Inhibition of Increase of HDR: ALT- Fbh1 siRNA/gRNA antirecombinant proteints HR, SSA, HR RTEL siRNA/gRNA PARI siRNA/gRNA Rap80 siRNA/gRNA miRNA miR-155-5p-antiMiR miR-155-3p-antiMiR miR-545-5p-antiMiR miR-545-3p-antiMiR miR-107-antiMiR miR-1255-A-antiMiR miR-1255-B1-antiMiR miR-1255-B2-antiMiR miR-148-5p-antiMiR miR-148-3p-antiMiR miR-193-5p-antiMiR miR-193-3p-antiMiR (E) Inhibition of Single Strand Increase HR Rad52 AID 651668 Annealing siRNA/gRNA ERCC1 NSC 130813, siRNA/gRNA XPF NSC 130813 siRNA/gRNA (F) Inhibition of Single Strand Increase HDR PARP Olaparib, AZD2281, Break Repair KU-0059436 Iniparib, BSI-201 BMN 673 Rucaparib, (AG014699, PF- 01367338) Veliparib, ABT-888 CEP 9722 INO-1001 MK 4827 BGB-290 E701, GPI21016 MP-124 LT-673 NMS-P118 XAV939 3-aminobenzamide XRCC1 siRNA/gRNA (H) Inhibition of MMR Increase HDR Msh2 siRNA/gRNA Cadmium (Cd(2+)) Msh3 siRNA/gRNA Msh6 siRNA/gRNA Cadmium (Cd(2+)) Mlh1 siRNA/gRNA Pms2 siRNA/gRNA (I) Chromatin modifier Increase HDR Ezh2 GSK343 inhibitors EPZ-6438 GSK2816126 SureCN6120847 EPZ005687 HDAC- Trichostatin A (TSA) Class I Sodium Butyrate (NaB) HDAC- Trichostatin A (TSA) Class II Sodium Butyrate (NaB) Setd2 siRNA/gRNA KDM4A/ siRNA/gRNA JMJD2A Cell Cycle arrest in G2 Increase HDR mostly CDk1 RO-3306 HR AZD 5438

VI.1 Homology-Directed Repair (HDR)

HDR is one of at least three repair pathways that act on double-stranded breaks (DSB). Which of the four pathways ultimately repairs a given DSB is influenced by a number of factors, including the degree of resection at the break. HDR typically acts when there has been significant resection at the DSB, forming at least one single stranded portion of DNA. The other three DSB repair pathways (canonical NHEJ, and alt-NHEJ) are discussed below. In addition to repairing DSBs, HDR (or a pathway sharing some of the same machinery) can also repair nicks when a nick is converted to a double strand break, e.g., after replication.

In some cases, the break is recognized by PARP1/2. PARP1/2 competes with Ku binding, and PARP1/2 binding favors engagement of the HDR machinery. Ku binding, in contrast, favors canonical NHEJ, as described below.

The DSB is also recognized by the MRN complex which contains MRE11, RAD50, and NBS1. MRE11 has 3′ to 5′ exonuclease activity and endonuclease activity. MrE11 can form a complex with RAD50, which results in the increase of exonuclease activity. The second subunit, NBS1, recruits ATM to the break. Resection at the break is initiated by the BRCA1-C complex. In this complex, CtBP-interacting protein (CtIP, also known as Retinoblastoma binding protein 8, carboxy terminal binding protein-interacting protein, or RBBP8) has endonuclease activity and interacts with BRCA1 and MRN. An initial step in the resection pathway may occur when BRCA1 displaces 53BP1-RIF1, which would otherwise push the break into the canonical NHEJ pathway. Once MRN and CtIP are assembled, endonucleolytic cleavage of the 5′ ends of the DSB creates short single-stranded 3′ overhangs. Next, resection enters the processive stage due to the activities of EXO1 exonuclease (which has 3′ to 5′ exonuclease activity), and the Dna2 endonuclease. (Dna2 possesses several enzymatic activities, including 5′ to 3′ exonuclease activity, ATPase activity, and helicase activity.) The helicases RECQl, BLM, WRN, RTS, RECQ4, and RECQ5 are human helicases involved in HDR. WRN has 3′ to 5′ helicase activity and exonuclease activity. BLM participates in replication and repair, unwinding both single strand and double stranded DNA in the 3′ to 5′ direction. During repair, BLM may also be involved in 5′ end resection. RecQ protein-like 4 (RECQ4 or RECQL4) has 3′ to 5′ helicase activity. RecQ helicase-like (RECQL, RECQl, or RECQL1) is a member of the RecQ helicase family and has 3′ to 5′ helicase activity. Together, these pro-resection components favor engagement of the HDR pathway.

Resection leads to the formation of single stranded DNA regions. These regions are bound and stabilized by RPA, a heterotrimer comprising RPA1, RPA2, and RPA3. An extended single strand can be repaired by the HDR pathway as discussed in this section, such as the SSA pathway which is discussed below. In the HDR pathway, the RPA heterotrimer undergoes post-translational modification, specifically PP4-dependent dephosphorylation of RPA2 and sumolyation of RPA1. RPA binding to the ssDNA generates a signal that has multiple consequence such as activation of the DNA damage response and ultimately the engagement of BRCA2. BRCA2 then acts to promote the RPA displacement and the consequential Rad51 loading onto the resected ends. CHK1 phosphorylates RAD51, allowing it to be recruited to the break. The Rad51 filament is a key factor involved in the search for homology and in promoting the D-loop invasion.

Repair can then progress via different DNA crossover intermediates, i.e., via the synthesis-dependent strand annealing (SDSA) pathway or by formation of double Holliday junctions (dHJs). Briefly, SDSA involves a DNA polymerase which lengthens the invading strand, and the RTEL helicase. When a dHJ is formed, the following machinery participates: BLM/TOPO III which dissolves the structure, an endonuclease such as FEN1, MUS81/EME1, or SLX1/SLX4 which cleaves the structure. (The FEN1 enzyme recognizes the specific DNA structure of 5′ overhanging flap structures that occur in DNA repair and replication, e.g., processes 5′ ends of Okazaki fragment during lagging strand synthesis. FEN1 may also possess 5′ to 3′ exonuclease activity on nicked or gapped double stranded DNA.)

In some embodiments, HDR results in physical integration of the template nucleic acid (or a part of it) into the genome as part of the repair process. In other embodiments, HDR does not result in physical integration of a part of the template nucleic acid into the genome.

FA (fanconi anemia) proteins may cause cells to favor HDR over canonical NHEJ.

In some embodiments, the methods herein involve up-regulating an HDR pathway(s). For instance, the methods may involve modulating (e.g., stimulating or overexpressing) a component (e.g., exactly one component, or one or more components, e.g., two or three components) of an HDR pathway, e.g., a component of Table VI.2 or VI.1(C). This component may be selected from, e.g., the group consisting of a PARP, PARP1, PARP2, MRN complex, MRE11, RAD50, NBS1, ATM, BRCA2, BRCA1, BRCA1 complex, BRCA1-C complex, BRCA1-B complex, CtIP, BRCA1, EX01, BLM, RPA complex, RPA1, RPA2, RPA3, PP4, or RAD51. In another embodiment, the up-regulator of HDR is a dominant negative CtIP. A dominant negative CtIP promotes resection in G1 phase.

In some embodiments, the HDR-enhancer molecule is a polypeptide of Table VI.2 or VI.1(C), or a polypeptide that comprises at least 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homology with, or differs by no more than 50, 40, 30, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, amino acid residues from a naturally occurring polypeptide of Table VI.2 or VI.1(C).

In some embodiments, the HDR enhancer comprises a PARP, PARP1, PARP2, member of a MRN complex, MRE11, RAD50, NBS1, BARD1, BRCA2, BRCA1, a member of a BRCA1 complex, a member of a BRCA1-C complex, a member of a BRCA1-B complex, CtIP, EX01, BLM, RECQl, WRN, RTS, RECQ5, RPA3, PP4, RAD51, BACH1, FANCJ, Topbp1, TOPO III, FEN1, MUS81, EME1, SLX1, SLX4, or a FA protein. In some embodiments, the HDR enhancer comprises at least 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homology with, or differs by no more than 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, amino acid residues from a naturally occurring a PARP, PARP1, PARP2, a member of a MRN complex, MRE11, RAD50, NBS1, BARD1, BRCA2, BRCA1, a member of a BRCA1 complex, a member of a BRCA1-C complex, a member of a BRCA1-B complex, CtIP, EX01, BLM, RECQl, WRN, RTS, RECQ5, RPA3, PP4, RAD51, BACH1, FANCJ, Topbp1, TOPO III, FEN1, MUS81, EME1, SLX1, SLX4, a FA protein, or one of the proteins described herein, e.g., in Table VI.2.

In another embodiment, an HDR enhancer may be an HDR-enhancing gRNA as described herein.

TABLE VI.2 Factors that promote HDR Factor Sequence a PARP — PARP1 MAESSDKLYRVEYAKSGRASCKKCSESIPKDSLRMAIMVQSPMFDGKVPHWYHFSCFWKVGHSIRHPDVE VDGFSELRWDDQQKVKKTAEAGGVTGKGQDGIGSKAEKTLGDFAAEYAKSNRSTCKGCMEKIEKGQVRLS KKMVDPEKPQLGMIDRWYHPGCFVKNREELGFRPEYSASQLKGFSLLATEDKEALKKQLPGVKSEGKRKG DEVDGVDEVAKKKSKKEKDKDSKLEKALKAQNDLIWNIKDELKKVCSTNDLKELLIFNKQQVPSGESAIL DRVADGMVFGALLPCEECSGQLVFKSDAYYCTGDVTAWTKCMVKTQTPNRKEWVTPKEFREISYLKKLKV KKQDRIFPPETSASVAATPPPSTASAPAAVNSSASADKPLSNMKILTLGKLSRNKDEVKAMIEKLGGKLT GTANKASLCISTKKEVEKMNKKMEEVKEANIRVVSEDFLQDVSASTKSLQELFLAHILSPWGAEVKAEPV EVVAPRGKSGAALSKKSKGQVKEEGINKSEKRMKLTLKGGAAVDPDSGLEHSAHVLEKGGKVFSATLGLV DIVKGTNSYYKLQLLEDDKENRYWIFRSWGRVGTVIGSNKLEQMPSKEDAIEHFMKLYEEKTGNAWHSKN FTKYPKKFYPLEIDYGQDEEAVKKLTVNPGTKSKLPKPVQDLIKMIFDVESMKKAMVEYEIDLQKMPLGK LSKRQIQAAYSILSEVQQAVSQGSSDSQILDLSNRFYTLIPHDFGMKKPPLLNNADSVQAKVEMLDNLLD IEVAYSLLRGGSDDSSKDPIDVNYEKLKTDIKVVDRDSEEAEIIRKYVKNTHATTHNAYDLEVIDIFKIE REGECQRYKPFKQLHNRRLLWHGSRTTNFAGILSQGLRIAPPEAPVTGYMFGKGIYFADMVSKSANYCHT SQGDPIGLILLGEVALGNMYELKHASHISKLPKGKHSVKGLGKTTPDPSANISLDGVDVPLGTGISSGVN DTSLLYNEYIVYDIAQVNLKYLLKLKFNFKTSLW (poly [ADP-ribose] polymerase 1 [Homo sapiens] CCDS 1554.1) (SEQ ID NO: 148) PARP2 MAARRRRSTGGGRARALNESKRVNNGNTAPEDSSPAKKTRRCQRQESKKMPVAGGKANKDRTEDKQDGMP GRSWASKRVSESVKALLLKGKAPVDPECTAKVGKAHVYCEGNDVYDVMLNQINLQFNNNKYYLIQLLEDD AQRNFSVWMRWGRVGKMGQHSLVACSGNLNKAKEIFQKKFLDKTKNNWEDREKFEKVPGKYDMLQMDYAT NTQDEEETKKEESLKSPLKPESQLDLRVQELIKLICNVQAMEEMMMEMKYNTKKAPLGKLTVAQIKAGYQ SLKKIEDCIRAGQHGRALMEACNEFYTRIPHDFGLRTPPLIRTQKELSEKIQLLEALGDIEIAIKLVKTE LQSPEHPLDQHYRNLHCALRPLDHESYEFKVISQYLQSTHAPTHSDYTMTLLDLFEVEKDGEKEAFREDL HNRMLLWHGSRMSNWVGILSHGLRIAPPEAPITGYMFGKGIYFADMSSKSANYCFASRLKNTGLLLLSEV ALGQCNELLEANPKAEGLLQGKHSTKGLGKMAPSSAHFVTLNGSTVPLGPASDTGILNPDGYTLNYNEYI VYNPNQVRMRYLLKVQFNFLQLW (poly [ADP-ribose] polymerase 2 isoform 2 [Homo sapiens] CCDS 41910.1) (SEQ ID NO: 149) MAARRRRSTGGGRARALNESKRVNNGNTAPEDSSPAKKTRRCQRQESKKMPVAGGKANKDRTEDKQDESV KALLLKGKAPVDPECTAKVGKAHVYCEGNDVYDVMLNQTNLQFNNNKYYLIQLLEDDAQRNFSVWMRWGR VGKMGQHSLVACSGNLNKAKEIFQKKFLDKTKNNWEDREKFEKVPGKYDMLQMDYATNTQDEEETKKEES LKSPLKPESQLDLRVQELIKLICNVQAMEEMMMEMKYNTKKAPLGKLTVAQIKAGYQSLKKIEDCIRAGQ HGRALMEACNEFYTRIPHDFGLRTPPLIRTQKELSEKIQLLEALGDIEIAIKLVKTELQSPEHPLDQHYR NLHCALRPLDHESYEFKVISQYLQSTHAPTHSDYTMTLLDLFEVEKDGEKEAFREDLHNRMLLWHGSRMS NWVGILSHGLRIAPPEAPITGYMFGKGIYFADMSSKSANYCFASRLKNTGLLLLSEVALGQCNELLEANP KAEGLLQGKHSTKGLGKMAPSSAHFVTLNGSTVPLGPASDTGILNPDGYTLNYNEYIVYNPNQVRMRYLL KVQFNFLQLW (poly [ADP-ribose] polymerase 2 isoform 1 [Homo sapiens] CCDS 45077.1) (SEQ ID NO: 150) MRN complex — MRE11 MSTADALDDENTFKILVATDIHLGFMEKDAVRGNDTFVTLDEILRLAQENEVDFILLGGD LFHENKPSRKTLHTCLELLRKYCMGDRPVQFEILSDQSVNFGFSKFPWVNYQDGNLNISI PVFSIHGNHDDPTGADALCALDILSCAGFVNHFGRSMSVEKIDISPVLLQKGSTKIALYG LGSIPDERLYRMFVNKKVTMLRPKEDENSWFNLFVIHQNRSKHGSTNFIPEQFLDDFIDL VIWGHEHECKIAPTKNEQQLFYISQPGSSVVTSLSPGEAVKKHVGLLRIKGRKMNMHKIP LHTVRQFFMEDIVLANHPDIFNPDNPKVTQAIQSFCLEKIEEMLENAERERLGNSHQPEK PLVRLRVDYSGGFEPFSVLRFSQKFVDRVANPKDIIHFFRHREQKEKTGEEINFGKLITK PSEGTTLRVEDLVKQYFQTAEKNVQLSLLTERGMGEAVQEFVDKEEKDAIEELVKYQLEK TQRFLKERHIDALEDKIDEEVRRFRETRQKNTNEEDDEVREAMTRARALRSQSEESASAF SADDLMSIDLAEQMANDSDDSISAATNKGRGRGRGRRGGRGQNSASRGGSQRGRAFKSTR QQPSRNVTTKNYSEVIEVDESDVEEDIFPTTSKTDQRWSSTSSSKIMSQSQVSKGVDFES SEDDDDDPFMNTSSLRRNRR (Mre11-isoform 2 CCDS 8298.1) (SEQ ID NO: 151) MSTADALDDENTFKILVATDIHLGFMEKDAVRGNDTFVTLDEILRLAQENEVDFILLGGD LFHENKPSRKTLHTCLELLRKYCMGDRPVQFEILSDQSVNFGFSKFPWVNYQDGNLNISI PVFSIHGNHDDPTGADALCALDILSCAGFVNHFGRSMSVEKIDISPVLLQKGSTKIALYG LGSIPDERLYRMFVNKKVTMLRPKEDENSWFNLFVIHQNRSKHGSTNFIPEQFLDDFIDL VIWGHEHECKIAPTKNEQQLFYISQPGSSVVTSLSPGEAVKKHVGLLRIKGRKMNMHKIP LHTVRQFFMEDIVLANHPDIFNPDNPKVTQAIQSFCLEKIEEMLENAERERLGNSHQPEK PLVRLRVDYSGGFEPFSVLRFSQKFVDRVANPKDIIHFFRHREQKEKTGEEINFGKLITK PSEGTTLRVEDLVKQYFQTAEKNVQLSLLTERGMGEAVQEFVDKEEKDAIEELVKYQLEK TQRFLKERHIDALEDKIDEEVRRFRETRQKNTNEEDDEVREAMTRARALRSQSEESASAF SADDLMSIDLAEQMANDSDDSISAATNKGRGRGRGRRGGRGQNSASRGGSQRGRADTGLE TSTRSRNSKTAVSASRNMSIIDAFKSTRQQPSRNVTTKNYSEVIEVDESDVEEDIFPTTS KTDQRWSSTSSSKIMSQSQVSKGVDFESSEDDDDDPFMNTSSLRRNRR (Mrell-isoform 1 CCDS8299.1) (SEQ ID NO: 152) RAD50 MSRIEKMSILGVRSFGIEDKDKQIITFFSPLTILVGPNGAGKTTIIECLKYICTGDFPPGTKGNTFVHDP KVAQETDVRAQIRLQFRDVNGELIAVQRSMVCTQKSKKTEFKTLEGVITRTKHGEKVSLSSKCAEIDREM ISSLGVSKAVLNNVIFCHQEDSNWPLSEGKALKQKFDEIFSATRYIKALETLRQVRQTQGQKVKEYQMEL KYLKQYKEKACEIRDQITSKEAQLTSSKEIVKSYENELDPLKNRLKEIEHNLSKIMKLDNEIKALDSRKK QMEKDNSELEEKMEKVFQGTDEQLNDLYHNHQRTVREKERKLVDCHRELEKLNKESRLLNQEKSELLVEQ GRLQLQADRHQEHIRARDSLIQSLATQLELDGFERGPFSERQIKNFHKLVRERQEGEAKTANQLMNDFAE KETLKQKQIDEIRDKKTGLGRIIELKSEILSKKQNELKNVKYELQQLEGSSDRILELDQELIKAERELSK AEKNSNVETLKMEVISLQNEKADLDRTLRKLDQEMEQLNHHTTTRTQMEMLTKDKADKDEQIRKIKSRHS DELTSLLGYFPNKKQLEDWLHSKSKEINQTRDRLAKLNKELASSEQNKNHINNELKRKEEQLSSYEDKLF DVCGSQDFESDLDRLKEEIEKSSKQRAMLAGATAVYSQFITQLTDENQSCCPVCQRVFQTEAELQEVISD LQSKLRLAPDKLKSTESELKKKEKRRDEMLGLVPMRQSIIDLKEKEIPELRNKLQNVNRDIQRLKNDIEE QETLLGTIMPEEESAKVCLTDVTIMERFQMELKDVERKIAQQAAKLQGIDLDRTVQQVNQEKQEKQHKLD TVSSKIELNRKLIQDQQEQIQHLKSTTNELKSEKLQISTNLQRRQQLEEQTVELSTEVQSLYREIKDAKE QVSPLETTLEKFQQEKEELINKKNTSNKIAQDKLNDIKEKVKNIHGYMKDIENYIQDGKDDYKKQKETEL NKVIAQLSECEKHKEKINEDMRLMRQDIDTQKIQERWLQDNLTLRKRNEELKEVEEERKQHLKEMGQMQV LQMKSEHQKLEENIDNIKRNHNLALGRQKGYEEEIIHFKKELREPQFRDAEEKYREMMIVMRTTELVNKD LDIYYKTLDQAIMKFHSMKMEEINKIIRDLWRSTYRGQDIEYIEIRSDADENVSASDKRRNYNYRVVMLK GDTALDMRGRCSAGQKVLASLIIRLALAETFCLNCGIIALDEPTTNLDRENIESLAHALVEIIKSRSQQR NFQLLVITHDEDFVELLGRSEYVEKFYRIKKNIDQCSEIVKCSVSSLGFNVH (Rad50 [Homo sapiens], CCDS 34233.1) (SEQ ID NO: 153) NBS1 MWKLLPAAGPAGGEPYRLLTGVEYVVGRKNCAILIENDQSISRNHAVLTANFSVTNLSQTDEIPVLTLKD NSKYGTFVNEEKMQNGFSRTLKSGDGITFGVFGSKFRIEYEPLVACSSCLDVSGKTALNQAILQLGGFTV NNWTEECTHLVMVSVKVTIKTICALICGRPIVKPEYFTEFLKAVESKKQPPQIESFYPPLDEPSIGSKNV DLSGRQERKQIFKGKTFIFLNAKQHKKLSSAVVFGGGEARLITEENEEEHNFFLAPGTCVVDTGITNSQT LIPDCQKKWIQSIMDMLQRQGLRPIPEAEIGLAVIFMTTKNYCDPQGHPSTGLKTTTPGPSLSQGVSVDE KLMPSAPVNTTTYVADTESEQADTWDLSERPKEIKVSKMEQKFRMLSQDAPTVKESCKTSSNNNSMVSNT LAKMRIPNYQLSPTKLPSINKSKDRASQQQQTNSIRNYFQPSTKKRERDEENQEMSSCKSARIETSCSLL EQTQPATPSLWKNKEQHLSENEPVDTNSDNNLFTDTDLKSIVKNSASKSHAAEKLRSNKKREMDDVAIED EVLEQLFKDTKPELEIDVKVQKQEEDVNVRKRPRMDIETNDTFSDEAVPESSKISQENEIGKKRELKEDS LWSAKEISNNDKLQDDSEMLPKKLLLTEFRSLVIKNSTSRNPSGINDDYGQLKNFKKFKKVTYPGAGKLP HIIGGSDLIAHHARKNTELEEWLRQEMEVQNQHAKEESLADDLFRYNPYLKRRR (NBS1 [Homo sapiens], CCDS 6249.1) (SEQ ID NO: 154) BARD1 MPDNRQPRNRQPRIRSGNEPRSAPAMEPDGRGAWAHSRAALDRLEKLLRCSRCTNILREPVCLGGCEHIF CSNCVSDCIGTGCPVCYTPAWIQDLKINRQLDSMIQLCSKLRNLLHDNELSDLKEDKPRKSLFNDAGNKK NSIKMWFSPRSKKVRYVVSKASVQTQPAIKKDASAQQDSYEFVSPSPPADVSERAKKASARSGKKQKKKT LAEINQKWNLEAEKEDGEFDSKEESKQKLVSFCSQPSVISSPQINGEIDLLASGSLTESECFGSLTEVSL PLAEQIESPDTKSRNEVVTPEKVCKNYLTSKKSLPLENNGKRGHHNRLSSPISKRCRTSILSTSGDFVKQ TVPSENIPLPECSSPPSCKRKVGGTSGRKNSNMSDEFISLSPGTPPSTLSSSSYRRVMSSPSAMKLLPNM AVKRNHRGETLLHIASIKGDIPSVEYLLQNGSDPNVKDHAGWTPLHEACNHGHLKVVELLLQHKALVNTT GYQNDSPLHDAAKNGHVDIVKLLLSYGASRNAVNIFGLRPVDYTDDESMKSLLLLPEKNESSSASHCSVM NTGQRRDGPLVLIGSGLSSEQQKMLSELAVILKAKKYTEFDSTVTHVVVPGDAVQSTLKCMLGILNGCWI LKFEWVKACLRRKVCEQEEKYEIPEGPRRSRLNREQLLPKLFDGCYFYLWGTFKHHPKDNLIKLVTAGGG QILSRKPKPDSDVTQTINTVAYHARPDSDQRFCTQYIIYEDLCNYHPERVRQGKVWKAPSSWFIDCVMSF ELLPLDS (BRCA1-associated RING domain protein 1 isoform 1 [Homo sapiens] CCDS 2397.1) (SEQ ID NO: 155) MPDNRQPRNRQPRIRSGNEPRSAPAMEPDGRGAWAHSRAALDRLEKLLRCSRCNCVSDCIGTGCPVCYTP AWIQDLKINRQLDSMIQLCSKLRNLLHDNELSDLKEDKPRKSLFNDAGNKKNSIKMWFSPRSKKVRYVVS KASVQTQPAIKKDASAQQDSYEFVSPSPPADVSERAKKASARSGKKQKKKTLAEINQKWNLEAEKEDGEF DSKEESKQKLVSFCSQPSVISSPQINGEIDLLASGSLTESECFGSLIEVSLPLAEQIESPDTKSRNEVVT PEKVCKNYLTSKKSLPLENNGKRGHHNRLSSPISKRCRTSILSTSGDFVKQTVPSENIPLPECSSPPSCK RKVGGTSGRKNSNMSDEFISLSPGTPPSTLSSSSYRRVMSSPSAMKLLPNMAVKRNHRGETLLHIASIKG DIPSVEYLLQNGSDPNVKDHAGWTPLHEACNHGHLKVVELLLQHKALVNTTGYQNDSPLHDAAKNGHVDI VKLLLSYGASRNAVNIFGLRPVDYTDDESMKSLLLLPEKNESSSASHCSVMNTGQRRDGPLVLIGSGLSS EQQKMLSELAVILKAKKYTEFDSTVIHVVVPGDAVQSTLKCMLGILNGCWILKFEWVKACLRRKVCEQEE KYEIPEGPRRSRLNREQLLPKLFDGCYFYLWGTFKHHPKDNLIKLVTAGGGQILSRKPKPDSDVTQTINT VAYHARPDSDQRFCTQYIIYEDLCNYHPERVRQGKVWKAPSSWFIDCVMSFELLPLDS (BRCA1- associated RING domain protein 1 isoform 2 [Homo sapiens] CCDS 74646.1) (SEQ ID NO: 156) MPDNRQPRNRQPRIRSGNEPRSAPAMEPDGRGAWAHSRAALDRLEKLLRCSRCINILREPVCLGGCEHIF CSNIFGLRPVDYTDDESMKSLLLLPEKNESSSASHCSVMNTGQRRDGPLVLIGSGLSSEQQKMLSELAVI LKAKKYTEFDSTVTHVVVPGDAVQSTLKCMLGILNGCWILKFEWVKACLRRKVCEQEEKYEIPEGPRRSR LNREQLLPKLFDGCYFYLWGTFKHHPKDNLIKLVTAGGGQILSRKPKPDSDVTQTINTVAYHARPDSDQR FCTQYIIYEDLCNYHPERVRQGKVWKAPSSWFIDCVMSFELLPLDS (BRCA1-associated RING domain protein 1 isoform 2 [Homo sapiens] CCDS 74647.1) (SEQ ID NO: 157) MPDNRQPRNRQPRIRSGNEPRSAPAMEPDGRGAWAHSRAALDRLEKLLRCSRCNIFGLRPVDYTDDESMK SLLLLPEKNESSSASHCSVMNTGQRRDGPLVLIGSGLSSEQQKMLSELAVILKAKKYTEFDSTVTHVVVP GDAVQSTLKCMLGILNGCWILKFEWVKACLRRKVCEQEEKYEIPEGPRRSRLNREQLLPKLFDGCYFYLW GTFKHHPKDNLIKLVTAGGGQILSRKPKPDSDVTQTINTVAYHARPDSDQRFCTQYIIYEDLCNYHPERV RQGKVWKAPSSWFIDCVMSFELLPLDS (BRCA1-associated RING domain protein 1 isoform 2 [Homo sapiens] CCDS 74645.1) (SEQ ID NO: 158) MPDNRQPRNRQPRIRSGNEPRSAPAMEPDGRGAWAHSRAALDRLEKLLRCSRCINILREPVCLGGCEHIF CSNCVSDCIGTGCPVCYTPAWIQDLKINRQLDSMIQLCSKLRNLLHDNELSGVKACLRRKVCEQEEKYEI PEGPRRSRLNREQLLPKLFDGCYFYLWGTFKHHPKDNLIKLVTAGGGQILSRKPKPDSDVTQTINTVAYH ARPDSDQRFCTQYIIYEDLCNYHPERVRQGKVWKAPSSWFIDCVMSFELLPLDS (BRCA1- associated RING domain protein 1 isoform 2 [Homo sapiens] CCDS 74648.1) (SEQ ID NO: 159) BRCA2 MPIGSKERPTFFEIFKTRCNKADLGPISLNWFEELSSEAPPYNSEPAEESEHKNNNYEPNLFKTPQRKPS YNQLASTPIIFKEQGLTLPLYQSPVKELDKFKLDLGRNVPNSRHKSLRTVKTKMDQADDVSCPLLNSCLS ESPVVLQCTHVIPQRDKSVVCGSLFHTPKFVKGRQTPKHISESLGAEVDPDMSWSSSLATPPILSSTVLI VRNEEASETVFPHDTTANVKSYFSNHDESLKKNDRFIASVTDSENTNQREAASHGFGKTSGNSFKVNSCK DHIGKSMPNVLEDEVYETVVDTSEEDSFSLCFSKCRTKNLQKVRTSKTRKKIFHEANADECEKSKNQVKE KYSFVSEVEPNDTDPLDSNVANQKPFESGSDKISKEVVPSLACEWSQLTLSGLNGAQMEKIPLLHISSCD QNISEKDLLDTENKRKKDFLTSENSLPRISSLPKSEKPLNEETVVNKRDEEQHLESHTDCILAVKQAISG TSPVASSFQGIKKSIFRIRESPKETFNASFSGHMTDPNFKKETEASESGLEIHTVCSQKEDSLCPNLIDN GSWPATTTQNSVALKNAGLISTLKKKTNKFIYAIHDETSYKGKKIPKDQKSELINCSAQFEANAFEAPLT FANADSGLLHSSVKRSCSQNDSEEPTLSLTSSFGTILRKCSRNETCSNNTVISQDLDYKEAKCNKEKLQL FITPEADSLSCLQEGQCENDPKSKKVSDIKEEVLAAACHPVQHSKVEYSDTDFQSQKSLLYDHENASTLI LTPTSKDVLSNLVMISRGKESYKMSDKLKGNNYESDVELTKNIPMEKNQDVCALNENYKNVELLPPEKYM RVASPSRKVQFNQNTNLRVIQKNQEETTSISKITVNPDSEELFSDNENNFVFQVANERNNLALGNTKELH ETDLTCVNEPIFKNSTMVLYGDTGDKQATQVSIKKDLVYVLAEENKNSVKQHIKMTLGQDLKSDISLNID KIPEKNNDYMNKWAGLLGPISNHSFGGSFRTASNKEIKLSEHNIKKSKMFFKDIEEQYPTSLACVEIVNT LALDNQKKLSKPQSINTVSAHLQSSVVVSDCKNSHITPQMLFSKQDFNSNHNLTPSQKAEITELSTILEE SGSQFEFTQFRKPSYILQKSTFEVPENQMTILKTTSEECRDADLHVIMNAPSIGQVDSSKQFEGTVEIKR KFAGLLKNDCNKSASGYLTDENEVGFRGFYSAHGTKLNVSTEALQKAVKLFSDIENISEETSAEVHPISL SSSKCHDSVVSMFKIENHNDKTVSEKNNKCQLILQNNIEMTTGTFVEEITENYKRNTENEDNKYTAASRN SHNLEFDGSDSSKNDTVCIHKDETDLLFTDQHNICLKLSGQFMKEGNTQIKEDLSDLTFLEVAKAQEACH GNTSNKEQLTATKTEQNIKDFETSDTFFQTASGKNISVAKESFNKIVNFFDQKPEELHNFSLNSELHSDI RKNKMDILSYEETDIVKHKILKESVPVGTGNQLVTFQGQPERDEKIKEPTLLGFHTASGKKVKIAKESLD KVKNLFDEKEQGTSEITSFSHQWAKTLKYREACKDLELACETIEITAAPKCKEMQNSLNNDKNLVSIETV VPPKLLSDNLCRQTENLKTSKSIFLKVKVHENVEKETAKSPATCYTNQSPYSVIENSALAFYTSCSRKTS VSQTSLLEAKKWLREGIFDGQPERINTADYVGNYLYENNSNSTIAENDKNHLSEKQDTYLSNSSMSNSYS YHSDEVYNDSGYLSKNKLDSGIEPVLKNVEDQKNTSFSKVISNVKDANAYPQTVNEDICVEELVTSSSPC KNKNAAIKLSISNSNNFEVGPPAFRIASGKIVCVSHETIKKVKDIFTDSFSKVIKENNENKSKICQTKIM AGCYEALDDSEDILHNSLDNDECSTHSHKVFADIQSEEILQHNQNMSGLEKVSKISPCDVSLETSDICKC SIGKLHKSVSSANTCGIFSTASGKSVQVSDASLQNARQVFSEIEDSTKQVFSKVLFKSNEHSDQLTREEN TAIRTPEHLISQKGFSYNVVNSSAFSGFSTASGKQVSILESSLHKVKGVLEEFDLIRTEHSLHYSPTSRQ NVSKILPRVDKRNPEHCVNSEMEKTCSKEFKLSNNLNVEGGSSENNHSIKVSPYLSQFQQDKQQLVLGTK VSLVENIHVLGKEQASPKNVKMEIGKTETFSDVPVKTNIEVCSTYSKDSENYFETEAVEIAKAFMEDDEL TDSKLPSHATHSLFTCPENEEMVLSNSRIGKRRGEPLILVGEPSIKRNLLNEFDRIIENQEKSLKASKST PDGTIKDRRLFMHHVSLEPITCVPFRTTKERQEIQNPNFTAPGQEFLSKSHLYEHLTLEKSSSNLAVSGH PFYQVSATRNEKMRHLITTGRPTKVFVPPFKTKSHFHRVEQCVRNINLEENRQKQNIDGHGSDDSKNKIN DNEIHQFNKNNSNQAVAVTFTKCEEEPLDLITSLQNARDIQDMRIKKKQRQRVFPQPGSLYLAKTSTLPR ISLKAAVGGQVPSACSHKQLYTYGVSKHCIKINSKNAESFQFHTEDYFGKESLWTGKGIQLADGGWLIPS NDGKAGKEEFYRALCDTPGVDPKLISRIWVYNHYRWIIWKLAAMECAFPKEFANRCLSPERVLLQLKYRY DTEIDRSRRSAIKKIMERDDTAAKTLVLCVSDIISLSANISETSSNKTSSADTQKVAIIELTDGWYAVKA QLDPPLLAVLKNGRLTVGQKIILHGAELVGSPDACTPLEAPESLMLKISANSTRPARWYTKLGFFPDPRP FPLPLSSLFSDGGNVGCVDVIIQRAYPIQWMEKTSSGLYIFRNEREEEKEAAKYVEAQQKRLEALFTKIQ EEFEEHEENTTKPYLPSRALTRQQVRALQDGAELYEAVKNAADPAYLEGYFSEEQLRALNNHRQMLNDKK QAQIQLEIRKAMESAEQKEQGLSRDVTTVWKLRIVSYSKKEKDSVILSIWRPSSDLYSLLTEGKRYRIYH LATSKSKSKSERANIQLAATKKTQYQQLPVSDEILFQIYQPREPLHFSKFLDPDFQPSCSEVDLIGFVVS VVKKTGLAPFVYLSDECYNLLAIKFWIDLNEDIIKPHMLIAASNLQWRPESKSGLLTLFAGDFSVFSASP KEGHFQETFNKMKNTVENIDILCNEAENKLMHILHANDPKWSTPTKDCTSGPYTAQIIPGTGNKLLMSSP NCEIYYQSPLSLCMAKRKSVSTPVSAQMTSKSCKGEKEIDDQKNCKKRRALDFLSRLPLPPPVSPICTFV SPAAQKAFQPPRSCGTKYETPIKKKELNSPQMTPFKKFNEISLLESNSIADEELALINTQALLSGSTGEK QFISVSESTRTAPTSSEDYLRLKRRCTTSLIKEQESSQASTEECEKNKQDTITTKKYI (breast cancer 2, early onset, isoform CRA_c [Homo sapiens] CCDS 9344.1) (SEQ ID NO: 160) BRCA1 MDLSALRVEEVQNVINAMQKILECPICLELIKEPVSTKCDHIFCKFCMLKLLNQKKGPSQCPLCKNDITK RSLQESTRFSQLVEELLKIICAFQLDTGLEYANSYNFAKKENNSPEHLKDEVSIIQSMGYRNRAKRLLQS EPENPSLQETSLSVQLSNLGTVRTLRTKQRIQPQKTSVYIELGSDSSEDTVNKATYCSVGDQELLQITPQ GTRDEISLDSAKKAACEFSETDVTNTEHHQPSNNDLNTTEKRAAERHPEKYQGSSVSNLHVEPCGTNTHA SSLQHENSSLLLTKDRMNVEKAEFCNKSKQPGLARSQHNRWAGSKETCNDRRTPSTEKKVDLNADPLCER KEWNKQKLPCSENPRDTEDVPWITLNSSIQKVNEWFSRSDELLGSDDSHDGESESNAKVADVLDVLNEVD EYSGSSEKIDLLASDPHEALICKSERVHSKSVESNIEDKIFGKTYRKKASLPNLSHVTENLIIGAFVTEP QIIQERPLTNKLKRKRRPTSGLHPEDFIKKADLAVQKTPEMINQGTNQTEQNGQVMNITNSGHENKTKGD SIQNEKNPNPIESLEKESAFKTKAEPISSSISNMELELNIHNSKAPKKNRLRRKSSTRHIHALELVVSRN LSPPNCTELQIDSCSSSEEIKKKKYNQMPVRHSRNLQLMEGKEPATGAKKSNKPNEQTSKRHDSDTFPEL KLTNAPGSFTKCSNTSELKEFVNPSLPREEKEEKLETVKVSNNAEDPKDLMLSGERVLQTERSVESSSIS LVPGTDYGTQESISLLEVSTLGKAKTEPNKCVSQCAAFENPKGLIHGCSKDNRNDTEGFKYPLGHEVNHS RETSIEMEESELDAQYLQNTFKVSKRQSFAPFSNPGNAEEECATFSAHSGSLKKQSPKVTFECEQKEENQ GKNESNIKPVQTVNITAGFPVVGQKDKPVDNAKCSIKGGSRFCLSSQFRGNETGLITPNKHGLLQNPYRI PPLFPIKSFVKTKCKKNLLEENFEEHSMSPEREMGNENIPSTVSTISRNNIRENVFKEASSSNINEVGSS TNEVGSSINEIGSSDENIQAELGRNRGPKLNAMLRLGVLQPEVYKQSLPGSNCKHPEIKKQEYEEVVQTV NTDFSPYLISDNLEQPMGSSHASQVCSETPDDLLDDGEIKEDTSFAENDIKESSAVFSKSVQKGELSRSP SPFTHTHLAQGYRRGAKKLESSEENLSSEDEELPCFQHLLFGKVNNIPSQSTRHSTVATECLSKNTEENL LSLKNSLNDCSNQVILAKASQEHHLSEETKCSASLFSSQCSELEDLTANTNTQDPFLIGSSKQMRHQSES QGVGLSDKELVSDDEERGTGLEENNQEEQSMDSNLGEAASGCESETSVSEDCSGLSSQSDILTTQQRDTM QHNLIKLQQEMAELEAVLEQHGSQPSNSYPSIISDSSALEDLRNPEQSTSEKAVLISQKSSEYPISQNPE GLSADKFEVSADSSTSKNKEPGVERSSPSKCPSLDDRWYMHSCSGSLQNRNYPSQEELIKVVDVEEQQLE ESGPHDLTETSYLPRQDLEGTPYLESGISLFSDDPESDPSEDRAPESARVGNIPSSTSALKVPQLKVAES AQSPAAAHTTDTAGYNAMEESVSREKPELTASTERVNKRMSMVVSGLTPEEFMLVYKFARKHHITLTNLI TEETTHVVMKTDAEFVCERTLKYFLGIAGGKWVVSYFWVIQSIKERKMLNEHDFEVRGDVVNGRNHQGPK RARESQDRKIFRGLEICCYGPFTNMPTDQLEWMVQLCGASVVKELSSFTLGTGVHPIVVVQPDAWTEDNG FHAIGQMCEAPVVIREWVLDSVALYQCQELDTYLIPQIPHSHY (breast cancer type 1 susceptibility protein isoform 1 [Homo sapiens] CCDS 11453.1) (SEQ ID NO: 161) MLKLLNQKKGPSQCPLCKNDITKRSLQESTRFSQLVEELLKIICAFQLDTGLEYANSYNFAKKENNSPEH LKDEVSIIQSMGYRNRAKRLLQSEPENPSLQETSLSVQLSNLGTVRTLRTKQRIQPQKTSVYIELGSDSS EDTVNKATYCSVGDQELLQITPQGTRDEISLDSAKKAACEFSETDVTNTEHHQPSNNDLNTTEKRAAERH PEKYQGSSVSNLHVEPCGTNTHASSLQHENSSLLLTKDRMNVEKAEFCNKSKQPGLARSQHNRWAGSKET CNDRRTPSTEKKVDLNADPLCERKEWNKQKLPCSENPRDTEDVPWITLNSSIQKVNEWFSRSDELLGSDD SHDGESESNAKVADVLDVLNEVDEYSGSSEKIDLLASDPHEALICKSERVHSKSVESNIEDKIFGKTYRK KASLPNLSHVTENLIIGAFVTEPQIIQERPLTNKLKRKRRPTSGLHPEDFIKKADLAVQKTPEMINQGTN QTEQNGQVMNITNSGHENKTKGDSIQNEKNPNPIESLEKESAFKTKAEPISSSISNMELELNIHNSKAPK KNRLRRKSSTRHIHALELVVSRNLSPPNCTELQIDSCSSSEEIKKKKYNQMPVRHSRNLQLMEGKEPATG AKKSNKPNEQTSKRHDSDTFPELKLTNAPGSFTKCSNTSELKEFVNPSLPREEKEEKLETVKVSNNAEDP KDLMLSGERVLQTERSVESSSISLVPGTDYGTQESISLLEVSTLGKAKTEPNKCVSQCAAFENPKGLIHG CSKDNRNDTEGFKYPLGHEVNHSRETSIEMEESELDAQYLQNTFKVSKRQSFAPFSNPGNAEEECATFSA HSGSLKKQSPKVIFECEQKEENQGKNESNIKPVQTVNITAGFPVVGQKDKPVDNAKCSIKGGSRFCLSSQ FRGNETGLITPNKHGLLQNPYRIPPLFPIKSFVKTKCKKNLLEENFEEHSMSPEREMGNENIPSTVSTIS RNNIRENVFKEASSSNINEVGSSTNEVGSSINEIGSSDENIQAELGRNRGPKLNAMLRLGVLQPEVYKQS LPGSNCKHPEIKKQEYEEVVQTVNTDFSPYLISDNLEQPMGSSHASQVCSETPDDLLDDGEIKEDTSFAE NDIKESSAVFSKSVQKGELSRSPSPFTHTHLAQGYRRGAKKLESSEENLSSEDEELPCFQHLLFGKVNNI PSQSTRHSTVATECLSKNTEENLLSLKNSLNDCSNQVILAKASQEHHLSEETKCSASLFSSQCSELEDLT ANTNTQDPFLIGSSKQMRHQSESQGVGLSDKELVSDDEERGTGLEENNQEEQSMDSNLGEAASGCESETS VSEDCSGLSSQSDILTTQQRDTMQHNLIKLQQEMAELEAVLEQHGSQPSNSYPSIISDSSALEDLRNPEQ STSEKAVLTSQKSSEYPISQNPEGLSADKFEVSADSSTSKNKEPGVERSSPSKCPSLDDRWYMHSCSGSL QNRNYPSQEELIKVVDVEEQQLEESGPHDLTETSYLPRQDLEGTPYLESGISLFSDDPESDPSEDRAPES ARVGNIPSSTSALKVPQLKVAESAQSPAAAHTTDTAGYNAMEESVSREKPELTASTERVNKRMSMVVSGL TPEEFMLVYKFARKHHITLTNLITEETTHVVMKTDAEFVCERTLKYFLGIAGGKWVVSYFWVTQSIKERK MLNEHDFEVRGDVVNGRNHQGPKRARESQDRKIFRGLEICCYGPFTNMPTDQLEWMVQLCGASVVKELSS FTLGTGVHPIVVVQPDAWTEDNGFHAIGQMCEAPVVTREWVLDSVALYQCQELDTYLIPQIPHSHY (breast cancer type 1 susceptibility protein isoform 2 [Homo sapiens] CCDS 11459.2) (SEQ ID NO: 162) MDLSALRVEEVQNVINAMQKILECPICLELIKEPVSTKCDHIFCKFCMLKLLNQKKGPSQCPLCKNDITK RSLQESTRFSQLVEELLKIICAFQLDTGLEYANSYNFAKKENNSPEHLKDEVSIIQSMGYRNRAKRLLQS EPENPSLQETSLSVQLSNLGTVRTLRTKQRIQPQKTSVYIELGSDSSEDTVNKATYCSVGDQELLQITPQ GTRDEISLDSAKKAACEFSETDVTNTEHHQPSNNDLNTTEKRAAERHPEKYQGSSVSNLHVEPCGTNTHA SSLQHENSSLLLTKDRMNVEKAEFCNKSKQPGLARSQHNRWAGSKETCNDRRTPSTEKKVDLNADPLCER KEWNKQKLPCSENPRDTEDVPWITLNSSIQKVNEWFSRSDELLGSDDSHDGESESNAKVADVLDVLNEVD EYSGSSEKIDLLASDPHEALICKSERVHSKSVESNIEDKIFGKTYRKKASLPNLSHVTENLIIGAFVTEP QIIQERPLTNKLKRKRRPTSGLHPEDFIKKADLAVQKTPEMINQGTNQTEQNGQVMNITNSGHENKTKGD SIQNEKNPNPIESLEKESAFKTKAEPISSSISNMELELNIHNSKAPKKNRLRRKSSTRHIHALELVVSRN LSPPNCTELQIDSCSSSEEIKKKKYNQMPVRHSRNLQLMEGKEPATGAKKSNKPNEQTSKRHDSDTFPEL KLTNAPGSFTKCSNTSELKEFVNPSLPREEKEEKLETVKVSNNAEDPKDLMLSGERVLQTERSVESSSIS LVPGTDYGTQESISLLEVSTLGKAKTEPNKCVSQCAAFENPKGLIHGCSKDNRNDTEGFKYPLGHEVNHS RETSIEMEESELDAQYLQNTFKVSKRQSFAPFSNPGNAEEECATFSAHSGSLKKQSPKVTFECEQKEENQ GKNESNIKPVQTVNITAGFPVVGQKDKPVDNAKCSIKGGSRFCLSSQFRGNETGLITPNKHGLLQNPYRI PPLFPIKSFVKTKCKKNLLEENFEEHSMSPEREMGNENIPSTVSTISRNNIRENVFKEASSSNINEVGSS TNEVGSSINEIGSSDENIQAELGRNRGPKLNAMLRLGVLQPEVYKQSLPGSNCKHPEIKKQEYEEVVQTV NTDFSPYLISDNLEQPMGSSHASQVCSETPDDLLDDGEIKEDTSFAENDIKESSAVFSKSVQKGELSRSP SPFTHTHLAQGYRRGAKKLESSEENLSSEDEELPCFQHLLFGKVNNIPSQSTRHSTVATECLSKNTEENL LSLKNSLNDCSNQVILAKASQEHHLSEETKCSASLFSSQCSELEDLTANTNTQDPFLIGSSKQMRHQSES QGVGLSDKELVSDDEERGTGLEENNQEEQSMDSNLGEAASGCESETSVSEDCSGLSSQSDILTTQQRDTM QHNLIKLQQEMAELEAVLEQHGSQPSNSYPSIISDSSALEDLRNPEQSTSEKDSHIHGQRNNSMFSKRPR EHISVLTSQKSSEYPISQNPEGLSADKFEVSADSSTSKNKEPGVERSSPSKCPSLDDRWYMHSCSGSLQN RNYPSQEELIKVVDVEEQQLEESGPHDLTETSYLPRQDLEGTPYLESGISLFSDDPESDPSEDRAPESAR VGNIPSSTSALKVPQLKVAESAQSPAAAHTTDTAGYNAMEESVSREKPELTASTERVNKRMSMVVSGLTP EEFMLVYKFARKHHITLTNLITEETTHVVMKTDAEFVCERTLKYFLGIAGGKWVVSYFWVTQSIKERKML NEHDFEVRGDVVNGRNHQGPKRARESQDRKIFRGLEICCYGPFTNMPTDQLEWMVQLCGASVVKELSSFT LGTGVHPIVVVQPDAWTEDNGFHAIGQMCEAPVVTREWVLDSVALYQCQELDTYLIPQIPHSHY (breast cancer type 1 susceptibility protein isoform 2 [Homo sapiens], CCDS 11456.2) (SEQ ID NO: 163) MDLSALRVEEVQNVINAMQKILECPICLELIKEPVSTKCDHIFCKFCMLKLLNQKKGPSQCPLCKNDITK RSLQESTRFSQLVEELLKIICAFQLDTGLEYANSYNFAKKENNSPEHLKDEVSIIQSMGYRNRAKRLLQS EPENPSLQETSLSVQLSNLGTVRTLRTKQRIQPQKTSVYIELGSDSSEDTVNKATYCSVGDQELLQITPQ GTRDEISLDSAKKAACEFSETDVTNTEHHQPSNNDLNTTEKRAAERHPEKYQGEAASGCESETSVSEDCS GLSSQSDILTIQQRDTMQHNLIKLQQEMAELEAVLEQHGSQPSNSYPSIISDSSALEDLRNPEQSTSEKV LTSQKSSEYPISQNPEGLSADKFEVSADSSTSKNKEPGVERSSPSKCPSLDDRWYMHSCSGSLQNRNYPS QEELIKVVDVEEQQLEESGPHDLTETSYLPRQDLEGTPYLESGISLFSDDPESDPSEDRAPESARVGNIP SSTSALKVPQLKVAESAQSPAAAHTTDTAGYNAMEESVSREKPELTASTERVNKRMSMVVSGLIPEEFML VYKFARKHHITLINLITEETTHVVMKTDAEFVCERTLKYFLGIAGGKWVVSYFWVIQSIKERKMLNEHDF EVRGDVVNGRNHQGPKRARESQDRKIFRGLEICCYGPFTNMPTDQLEWMVQLCGASVVKELSSFTLGTGV HPIVVVQPDAWTEDNGFHAIGQMCEAPVVIREWVLDSVALYQCQELDTYLIPQIPHSHY (breast cancer type 1 susceptibility protein isoform 2 [Homo sapiens] CCDS 11454.2) (SEQ ID NO: 164) MDLSALRVEEVQNVINAMQKILECPICLELIKEPVSTKCDHIFCKFCMLKLLNQKKGPSQCPLCKNDITK RSLQESTRFSQLVEELLKIICAFQLDTGLEYANSYNFAKKENNSPEHLKDEVSIIQSMGYRNRAKRLLQS EPENPSLQETSLSVQLSNLGTVRTLRTKQRIQPQKTSVYIELGSDSSEDTVNKATYCSVGDQELLQITPQ GTRDEISLDSAKKAACEFSETDVTNTEHHQPSNNDLNTTEKRAAERHPEKYQGEAASGCESETSVSEDCS GLSSQSDILTTQQRDTMQHNLIKLQQEMAELEAVLEQHGSQPSNSYPSIISDSSALEDLRNPEQSTSEKV LTSQKSSEYPISQNPEGLSADKFEVSADSSTSKNKEPGVERSSPSKCPSLDDRWYMHSCSGSLQNRNYPS QEELIKVVDVEEQQLEESGPHDLTETSYLPRQDLEGTPYLESGISLFSDDPESDPSEDRAPESARVGNIP SSTSALKVPQLKVAESAQSPAAAHTTDTAGYNAMEESVSREKPELTASTERVNKRMSMVVSGLIPEEFML VYKFARKHHITLINLITEETTHVVMKTDAEFVCERTLKYFLGIAGGKWVVSYFWVIQSIKERKMLNEHDF EVRGDVVNGRNHQGPKRARESQDRKIFRGLEICCYGPFTNMPTGCPPNCGCAARCLDRGQWLPCNWADV (breast cancer type 1 susceptibility protein isoform 2 [Homo sapiens] CCDS 11455.2) (SEQ ID NO: 165) BRCA1-C complex — BRCA1-B complex — CtIP MNISGSSCGSPNSADTSSDFKDLWTKLKECHDREVQGLQVKVTKLKQERILDAQRLEEFF TKNQQLREQQKVLHETIKVLEDRLRAGLCDRCAVTEEHMRKKQQEFENIRQQNLKLITEL MNERNTLQEENKKLSEQLQQKIENDQQHQAAELECEEDVIPDSPITAFSFSGVNRLRRKE NPHVRYIEQTHIKLEHSVCANEMRKVSKSSTHPQHNPNENEILVADTYDQSQSPMAKAHG TSSYTPDKSSFNLATVVAETLGLGVQEESETQGPMSPLGDELYHCLEGNHKKQPFEESTR NTEDSLRFSDSTSKTPPQEELPTRVSSPVFGATSSIKSGLDLNTSLSPSLLQPGKKKHLK TLPFSNTCISRLEKTRSKSEDSALFTHHSLGSEVNKIIIQSSNKQILINKNISESLGEQN RTEYGKDSNTDKHLEPLKSLGGRTSKRKKTEEESEHEVSCPQASFDKENAFPFPMDNQFS MNGDCVMDKPLDLSDRFSAIQRQEKSQGSETSKNKFRQVTLYEALKTIPKGFSSSRKASD GNCTLPKDSPGEPCSQECIILQPLNKCSPDNKPSLQIKEENAVFKIPLRPRESLETENVL DDIKSAGSHEPIKIQTRSDHGGCELASVLQLNPCRTGKIKSLQNNQDVSFENIQWSIDPG ADLSQYKMDVTVIDTKDGSQSKLGGETVDMDCTLVSETVLLKMKKQEQKGEKSSNEERKM NDSLEDMFDRTTHEEYESCLADSFSQAADEEEELSTATKKLHTHGDKQDKVKQKAFVEPY FKGDERETSLQNFPHIEVVRKKEERRKLLGHTCKECEIYYADMPAEEREKKLASCSRHRF RYIPPNTPENFWEVGFPSTQTCMERGYIKEDLDPCPRPKRRQPYNAIFSPKGKEQKT (CtIP- isoform 1 CCDS 11875.1) (SEQ ID NO: 166) MNISGSSCGSPNSADTSSDFKDLWTKLKECHDREVQGLQVKVTKLKQERILDAQRLEEFF TKNQQLREQQKVLHETIKVLEDRLRAGLCDRCAVTEEHMRKKQQEFENIRQQNLKLITEL MNERNTLQEENKKLSEQLQQKIENDQQHQAAELECEEDVIPDSPITAFSFSGVNRLRRKE NPHVRYIEQTHTKLEHSVCANEMRKVSKSSTHPQHNPNENEILVADTYDQSQSPMAKAHG TSSYTPDKSSFNLATVVAETLGLGVQEESETQGPMSPLGDELYHCLEGNHKKQPFEESTR NTEDSLRFSDSTSKTPPQEELPTRVSSPVFGATSSIKSGLDLNTSLSPSLLQPGKKKHLK TLPFSNTCISRLEKTRSKSEDSALFTHHSLGSEVNKIIIQSSNKQILINKNISESLGEQN RTEYGKDSNTDKHLEPLKSLGGRTSKRKKTEEESEHEVSCPQASFDKENAFPFPMDNQFS MNGDCVMDKPLDLSDRFSAIQRQEKSQGSETSKNKFRQVTLYEALKTIPKGFSSSRKASD GNCTLPKDSPGEPCSQECIILQPLNKCSPDNKPSLQIKEENAVFKIPLRPRESLETENVL DDIKSAGSHEPIKIQTRSDHGGCELASVLQLNPCRTGKIKSLQNNQDVSFENIQWSIDPG ADLSQYKMDVTVIDTKDGSQSKLGGETVDMDCTLVSETVLLKMKKQEQKGEKSSNEERKM NDSLEDMFDRTTHEEYESCLADSFSQAADEEEELSTATKKLHTHGDKQDKVKQKAFVEPY FKGDESIMQICQQKKEKRNWLPAQDTDSATFHPTHQRIFGKLVFLPLRLVWKEVILRKIL ILVLVQKDVSLTTQYFLQKARSRRHRR (CtIP-isoform 2 CCDS 11874.1) (SEQ ID NO: 167) Dominant Negative CtIP ATGAACATCTTGGGAAGCAGCTGTGGAAGCCCTAACTCTGCAGATACATCTAGTGACTTTAAGGACCTTT (S327E, T847E, or GGACAAAACTAAAAGAATGTCATGATAGAGAAGTACAAGGTTTACAAGTAAAAGTAACCAAGCTAAAACA S327E + T847E) GGAACGAATCTTAGATGCACAAAGACTAGAAGAATTCTTCACCAAAAATCAACAGCTGAGGGAACAGCAG AAAGTCCTTCATGAAACCATTAAAGTTTTAGAAGATCGGTTAAGAGCAGGCTTATGTGATCGCTGTGCAG TAACTGAAGAACATATGCGGAAAAAACAGCAAGAGTTTGAAAATATCCGGCAGCAGAATCTTAAACTTAT TACAGAACTTATGAATGAAAGGAATACTCTACAGGAAGAAAATAAAAAGCTTTCTGAACAACTCCAGCAG AAAATTGAGAATGATCAACAGCATCAAGCAGCTGAGCTTGAATGTGAGGAAGACGTTATTCCAGATTCAC CGATAACAGCCTTCTCATTTTCTGGCGTTAACCGGCTACGAAGAAAGGAGAACCCCCATGTCCGATACAT AGAACAAACACATACTAAATTGGAGCACTCTGTGTGTGCAAATGAAATGAGAAAAGTTTCCAAGTCTTCA ACTCATCCACAACATAATCCTAATGAAAATGAAATTCTAGTAGCTGACACTTATGACCAAAGTCAATCTC CAATGGCCAAAGCACATGGAACAAGCAGCTATACCCCTGATAAGTCATCTTTTAATTTAGCTACAGTTGT TGCTGAAACACTTGGACTTGGTGTTCAAGAAGAATCTGAAACTCAAGGTCCCATGAGCCCCCTTGGTGAT GAGCTCTACCACTGTCTGGAAGGAAATCACAAGAAACAGCCTTTTGAGGAATCTACAAGAAATACTGAAG ATAGTTTAAGATTTTCAGATTCTACTTCAAAGACTCCTCCTCAAGAAGAATTACCTACTCGAGTGTCATC TCCTGTATTTGGAGCTACCTCTAGTATCAAAAGTGGTTTAGATTTGAATACAAGTTTGTCCCCTTCTCTT TTACAGCCTGGGAAAAAAAAACATCTGAAAACACTCCCTTTTAGCAACACTTGTATATCTAGATTAGAAA AAACTAGATCAAAATCTGAAGATAGTGCCCTTTTCACACATCACAGTCTTGGGTCTGAAGTGAACAAGAT CATTATCCAGTCATCTAATAAACAGATACTTATAAATAAAAATATAAGTGAATCCCTAGGTGAACAGAAT AGGACTGAGTACGGTAAAGATTCTAACACTGATAAACATTTGGAGCCCCTGAAATCATTGGGAGGCCGAA CATCCAAAAGGAAGAAAACTGAGGAAGAAAGTGAACATGAAGTAAGCTGCCCCCAAGCTTCTTTTGATAA AGAAAATGCTTTCCCTTTTCCAATGGATAATCAGTTTTCCATGAATGGAGACTGTGTGATGGATAAACCT CTGGATCTGTCTGATCGATTTTCAGCTATTCAGCGTCAAGAGAAAAGCCAAGGAAGTGAGACTTCTAAAA ACAAATTTAGGCAAGTGACTCTTTATGAGGCTTTGAAGACCATTCCAAAGGGCTTTTCCTCAAGCCGTAA GGCCTCAGATGGCAACTGCACGTTGCCCAAAGATTCCCCAGGGGAGCCCTGTTCACAGGAATGCATCATC CTTCAGCCCTTGAATAAATGCTCTCCAGACAATAAACCATCATTACAAATAAAAGAAGAAAATGCTGTCT TTAAAATTCCTCTACGTCCACGTGAAAGTTTGGAGACTGAGAATGTTTTAGATGACATAAAGAGTGCTGG TTCTCATGAGCCAATAAAAATACAAACCAGGTCAGACCATGGAGGATGTGAACTTGCATCAGTTCTTCAG TTAAATCCATGTAGAACTGGTAAAATAAAGTCTCTACAAAACAACCAAGATGTATCCTTTGAAAATATCC AGTGGAGTATAGATCCGGGAGCAGACCTTTCTCAGTATAAAATGGATGTTACTGTAATAGATACAAAGGA TGGCAGTCAGTCAAAATTAGGAGGAGAGACAGTGGACATGGACTGTACATTGGTTAGTGAAACCGTTCTC TTAAAAATGAAGAAGCAAGAGCAGAAGGGAGAAAAAAGTTCAAATGAAGAAAGAAAAATGAATGATAGCT TGGAAGATATGTTTGATCGGACAACACATGAAGAGTATGAATCCTGTTTGGCAGACAGTTTCTCCCAAGC AGCAGATGAAGAGGAGGAATTGTCTACTGCCACAAAGAAACTACACACTCATGGTGATAAACAAGACAAA GTCAAGCAGAAAGCGTTTGTGGAGCCGTATTTTAAAGGTGATGAAAGAGAGACTAGCTTGCAAAATTTTC CTCATATTGAGGTGGTTCGGAAAAAAGAGGAGAGAAGAAAACTGCTTGGGCACACGTGTAAGGAATGTGA AATTTATTATGCAGATATGCCAGCAGAAGAAAGAGAAAAGAAATTGGCTTCCTGCTCAAGACACCGATTC CGCTACATTCCACCCAACACACCAGAGAATTTTTGGGAAGTTGGTTTTCCTTCCACTCAGACTTGTATGG AAAGAGGTTATATTAAGGAAGATCTTGATCCTTGTCCTCGTCCAAAAAGACGTCAGCCTTACAACGCAAT ATTTTCTCCAAAAGGCAAGGAGCAGAAGACATAA 327, 847 (SEQ ID NO: 168) MNILGSSCGSPNSADTSSDFKDLWTKLKECHDREVQGLQVKVTKLKQERILDAQRLEEFFTKNQQLREQQ KVLHETIKVLEDRLRAGLCDRCAVTEEHMRKKQQEFENIRQQNLKLITELMNERNTLQEENKKLSEQLQQ KIENDQQHQAAELECEEDVIPDSPITAFSFSGVNRLRRKENPHVRYIEQTHTKLEHSVCANEMRKVSKSS THPQHNPNENEILVADTYDQSQSPMAKAHGTSSYTPDKSSFNLATVVAETLGLGVQEESETQGPMSPLGD ELYHCLEGNHKKQPFEESTRNTEDSLRFSDSTSKTPPQEELPTRVSSPVFGATSSIKSGLDLNTSLSPSL LQPGKKKHLKTLPFSNTCISRLEKTRSKSEDSALFTHHSLGSEVNKIIIQSSNKQILINKNISESLGEQN RTEYGKDSNIDKHLEPLKSLGGRISKRKKTEEESEHEVSCPQASFDKENAFPFPMDNQFSMNGDCVMDKP LDLSDRFSAIQRQEKSQGSETSKNKFRQVTLYEALKTIPKGFSSSRKASDGNCTLPKDSPGEPCSQECII LQPLNKCSPDNKPSLQIKEENAVFKIPLRPRESLETENVLDDIKSAGSHEPIKIQTRSDHGGCELASVLQ LNPCRTGKIKSLQNNQDVSFENIQWSIDPGADLSQYKMDVTVIDTKDGSQSKLGGETVDMDCTLVSETVL LKMKKQEQKGEKSSNEERKMNDSLEDMFDRTTHEEYESCLADSFSQAADEEEELSTATKKLHTHGDKQDK VKQKAFVEPYFKGDERETSLQNFPHIEVVRKKEERRKLLGHICKECEIYYADMPAEEREKKLASCSRHRF RYIPPNTPENFWEVGFPSTQTCMERGYIKEDLDPCPRPKRRQPYNAIFSPKGKEQKTDYKDHDGDYKDHD I** (SEQ ID NO: 169) ATGAACATCTTGGGAAGCAGCTGTGGAAGCCCTAACTCTGCAGATACATCTAGTGACTTTAAGGACCTTT GGACAAAACTAAAAGAATGTCATGATAGAGAAGTACAAGGTTTACAAGTAAAAGTAACCAAGCTAAAACA GGAACGAATCTTAGATGCACAAAGACTAGAAGAATTCTTCACCAAAAATCAACAGCTGAGGGAACAGCAG AAAGTCCTTCATGAAACCATTAAAGTTTTAGAAGATCGGTTAAGAGCAGGCTTATGTGATCGCTGTGCAG TAACTGAAGAACATATGCGGAAAAAACAGCAAGAGTTTGAAAATATCCGGCAGCAGAATCTTAAACTTAT TACAGAACTTATGAATGAAAGGAATACTCTACAGGAAGAAAATAAAAAGCTTTCTGAACAACTCCAGCAG AAAATTGAGAATGATCAACAGCATCAAGCAGCTGAGCTTGAATGTGAGGAAGACGTTATTCCAGATTCAC CGATAACAGCCTTCTCATTTTCTGGCGTTAACCGGCTACGAAGAAAGGAGAACCCCCATGTCCGATACAT AGAACAAACACATACTAAATTGGAGCACTCTGTGTGTGCAAATGAAATGAGAAAAGTTTCCAAGTCTTCA ACTCATCCACAACATAATCCTAATGAAAATGAAATTCTAGTAGCTGACACTTATGACCAAAGTCAATCTC CAATGGCCAAAGCACATGGAACAAGCAGCTATACCCCTGATAAGTCATCTTTTAATTTAGCTACAGTTGT TGCTGAAACACTTGGACTTGGTGTTCAAGAAGAATCTGAAACTCAAGGTCCCATGAGCCCCCTTGGTGAT GAGCTCTACCACTGTCTGGAAGGAAATCACAAGAAACAGCCTTTTGAGGAATCTACAAGAAATACTGAAG ATAGTTTAAGATTTTCAGATTCTACTTCAAAGACTCCTCCTCAAGAAGAATTACCTACTCGAGTGTCATC TCCTGTATTTGGAGCTACCTCTAGTATCAAAAGTGGTTTAGATTTGAATACAAGTTTGTCCCCTTCTCTT TTACAGCCTGGGAAAAAAAAACATCTGAAAACACTCCCTTTTAGCAACACTTGTATATCTAGATTAGAAA AAACTAGATCAAAATCTGAAGATAGTGCCCTTTTCACACATCACAGTCTTGGGTCTGAAGTGAACAAGAT CATTATCCAGTCATCTAATAAACAGATACTTATAAATAAAAATATAAGTGAATCCCTAGGTGAACAGAAT AGGACTGAGTACGGTAAAGATTCTAACACTGATAAACATTTGGAGCCCCTGAAATCATTGGGAGGCCGAA CATCCAAAAGGAAGAAAACTGAGGAAGAAAGTGAACATGAAGTAAGCTGCCCCCAAGCTTCTTTTGATAA AGAAAATGCTTTCCCTTTTCCAATGGATAATCAGTTTTCCATGAATGGAGACTGTGTGATGGATAAACCT CTGGATCTGTCTGATCGATTTTCAGCTATTCAGCGTCAAGAGAAAAGCCAAGGAAGTGAGACTTCTAAAA ACAAATTTAGGCAAGTGACTCTTTATGAGGCTTTGAAGACCATTCCAAAGGGCTTTTCCTCAAGCCGTAA GGCCTCAGATGGCAACTGCACGTTGCCCAAAGATTCCCCAGGGGAGCCCTGTTCACAGGAATGCATCATC CTTCAGCCCTTGAATAAATGCTCTCCAGACAATAAACCATCATTACAAATAAAAGAAGAAAATGCTGTCT TTAAAATTCCTCTACGTCCACGTGAAAGTTTGGAGACTGAGAATGTTTTAGATGACATAAAGAGTGCTGG TTCTCATGAGCCAATAAAAATACAAACCAGGTCAGACCATGGAGGATGTGAACTTGCATCAGTTCTTCAG TTAAATCCATGTAGAACTGGTAAAATAAAGTCTCTACAAAACAACCAAGATGTATCCTTTGAAAATATCC AGTGGAGTATAGATCCGGGAGCAGACCTTTCTCAGTATAAAATGGATGTTACTGTAATAGATACAAAGGA TGGCAGTCAGTCAAAATTAGGAGGAGAGACAGTGGACATGGACTGTACATTGGTTAGTGAAACCGTTCTC TTAAAAATGAAGAAGCAAGAGCAGAAGGGAGAAAAAAGTTCAAATGAAGAAAGAAAAATGAATGATAGCT TGGAAGATATGTTTGATCGGACAACACATGAAGAGTATGAATCCTGTTTGGCAGACAGTTTCTCCCAAGC AGCAGATGAAGAGGAGGAATTGTCTACTGCCACAAAGAAACTACACACTCATGGTGATAAACAAGACAAA GTCAAGCAGAAAGCGTTTGTGGAGCCGTATTTTAAAGGTGATGAAAGAGAGACTAGCTTGCAAAATTTTC CTCATATTGAGGTGGTTCGGAAAAAAGAGGAGAGAAGAAAACTGCTTGGGCACACGTGTAAGGAATGTGA AATTTATTATGCAGATATGCCAGCAGAAGAAAGAGAAAAGAAATTGGCTTCCTGCTCAAGACACCGATTC CGCTACATTCCACCCAACGAACCAGAGAATTTTTGGGAAGTTGGTTTTCCTTCCACTCAGACTTGTATGG AAAGAGGTTATATTAAGGAAGATCTTGATCCTTGTCCTCGTCCAAAAAGACGTCAGCCTTACAACGCAAT ATTTTCTCCAAAAGGCAAGGAGCAGAAGACATAA (SEQ ID NO: 170) MNILGSSCGSPNSADTSSDFKDLWTKLKECHDREVQGLQVKVTKLKQERILDAQRLEEFFTKNQQLREQQ KVLHETIKVLEDRLRAGLCDRCAVTEEHMRKKQQEFENIRQQNLKLITELMNERNTLQEENKKLSEQLQQ KIENDQQHQAAELECEEDVIPDSPITAFSFSGVNRLRRKENPHVRYIEQTHTKLEHSVCANEMRKVSKSS THPQHNPNENEILVADTYDQSQSPMAKAHGTSSYTPDKSSFNLATVVAETLGLGVQEESETQGPMSPLGD ELYHCLEGNHKKQPFEESTRNTEDSLRFSDSTSKTPPQEELPTRVSSPVFGATSSIKSGLDLNTSLSPSL LQPGKKKHLKTLPFSNTCISRLEKTRSKSEDSALFTHHSLGSEVNKIIIQSSNKQILINKNISESLGEQN RTEYGKDSNTDKHLEPLKSLGGRTSKRKKTEEESEHEVSCPQASFDKENAFPFPMDNQFSMNGDCVMDKP LDLSDRFSAIQRQEKSQGSETSKNKFRQVTLYEALKTIPKGFSSSRKASDGNCTLPKDSPGEPCSQECII LQPLNKCSPDNKPSLQIKEENAVFKIPLRPRESLETENVLDDIKSAGSHEPIKIQTRSDHGGCELASVLQ LNPCRTGKIKSLQNNQDVSFENIQWSIDPGADLSQYKMDVTVIDTKDGSQSKLGGETVDMDCTLVSETVL LKMKKQEQKGEKSSNEERKMNDSLEDMFDRTTHEEYESCLADSFSQAADEEEELSTATKKLHTHGDKQDK VKQKAFVEPYFKGDERETSLQNFPHIEVVRKKEERRKLLGHTCKECEIYYADMPAEEREKKLASCSRHRF RYIPPNEPENFWEVGFPSTQTCMERGYIKEDLDPCPRPKRRQPYNAIFSPKGKEQKTDYKDHDGDYKDHD I* (SEQ ID NO: 171) EXO1 MGIQGLLQFIKEASEPIHVRKYKGQVVAVDTYCWLHKGAIACAEKLAKGEPTDRYVGFCM KFVNMLLSHGIKPILVFDGCTLPSKKEVERSRRERRQANLLKGKQLLREGKVSEARECFT RSINITHAMAHKVIKAARSQGVDCLVAPYEADAQLAYLNKAGIVQATITEDSDLLAFGCK KVILKMDQFGNGLEIDQARLGMCRQLGDVFTEEKFRYMCILSGCDYLSSLRGIGLAKACK VLRLANNPDIVKVIKKIGHYLKMNITVPEDYINGFIRANNTFLYQLVFDPIKRKLIPLNA YEDDVDPETLSYAGQYVDDSIALQIALGNKDINTFEQIDDYNPDTAMPAHSRSHSWDDKT CQKSANVSSIWHRNYSPRPESGTVSDAPQLKENPSTVGVERVISTKGLNLPRKSSIVKRP RSAELSEDDLLSQYSLSFTKKTKKNSSEGNKSLSFSEVFVPDLVNGPTNKKSVSTPPRTR NKFATFLQRKNEESGAVVVPGTRSRFFCSSDSTDCVSNKVSIQPLDETAVTDKENNLHES EYGDQEGKRLVDTDVARNSSDDIPNNHIPGDHIPDKATVFTDEESYSFESSKFTRTISPP TLGTLRSCFSWSGGLGDFSRTPSPSPSTALQQFRRKSDSPTSLPENNMSDVSQLKSEESS DDESHPLREEACSSQSQESGEFSLQSSNASKLSQCSSKDSDSEESDCNIKLLDSQSDQTS KLRLSHFSKKDTPLRNKVPGLYKSSSADSLSTTKIKPLGPARASGLSKKPASIQKRKHHN AENKPGLQIKLNELWKNFGFKKF (Exo1 Isoform 1 CCDS 44336.1) (SEQ ID NO: 172) MGIQGLLQFIKEASEPIHVRKYKGQVVAVDTYCWLHKGAIACAEKLAKGEPTDRYVGFCM KFVNMLLSHGIKPILVFDGCTLPSKKEVERSRRERRQANLLKGKQLLREGKVSEARECFT RSINITHAMAHKVIKAARSQGVDCLVAPYEADAQLAYLNKAGIVQATITEDSDLLAFGCK KVILKMDQFGNGLEIDQARLGMCRQLGDVFTEEKFRYMCILSGCDYLSSLRGIGLAKACK VLRLANNPDIVKVIKKIGHYLKMNITVPEDYINGFIRANNTFLYQLVFDPIKRKLIPLNA YEDDVDPETLSYAGQYVDDSIALQIALGNKDINTFEQIDDYNPDTAMPAHSRSHSWDDKT CQKSANVSSIWHRNYSPRPESGTVSDAPQLKENPSTVGVERVISTKGLNLPRKSSIVKRP RSAELSEDDLLSQYSLSFTKKTKKNSSEGNKSLSFSEVFVPDLVNGPTNKKSVSTPPRTR NKFATFLQRKNEESGAVVVPGTRSRFFCSSDSTDCVSNKVSIQPLDETAVTDKENNLHES EYGDQEGKRLVDTDVARNSSDDIPNNHIPGDHIPDKATVFTDEESYSFESSKFTRTISPP TLGTLRSCFSWSGGLGDFSRTPSPSPSTALQQFRRKSDSPTSLPENNMSDVSQLKSEESS DDESHPLREEACSSQSQESGEFSLQSSNASKLSQCSSKDSDSEESDCNIKLLDSQSDQTS KLRLSHFSKKDTPLRNKVPGLYKSSSADSLSTTKIKPLGPARASGLSKKPASIQKRKHHN AENKPGLQIKLNELWKNFGFKKDSEKLPPCKKPLSPVRDNIQLTPEAEEDIFNKPECGRV QRAIFQ (Exo1 Isoform 2 CCDS 1620.1) (SEQ ID NO: 173) BLM MAAVPQNNLQEQLERHSARTLNNKLSLSKPKFSGFIFKKKISSDNNVSVINVSVAKTPVL RNKDVNVIEDFSFSEPLPNTINQQRVKDFFKNAPAGQETQRGGSKSLLPDFLQTPKEVVC TTQNTPTVKKSRDTALKKLEFSSSPDSLSTINDWDDMDDFDTSETSKSFVTPPQSHFVRV STAQKSKKGKRNFFKAQLYTTNTVKTDLPPPSSESEQIDLTEEQKDDSEWLSSDVICIDD GPIAEVHINEDAQESDSLKTHLEDERDNSEKKKNLEEAELHSTEKVPCIEFDDDDYDTDF VPPSPEEIISASSSSSKCLSTLKDLDTSDRKEDVLSTSKDLLSKPEKMSMQELNPETSTD CDARQISLQQQLIHVMEHICKLIDTIPDDKLKLLDCGNELLQQRNIRRKLLTEVDFNKSD ASLLGSLWRYRPDSLDGPMEGDSCPTGNSMKELNFSHLPSNSVSPGDCLLTTTLGKTGFS ATRKNLFERPLFNTHLQKSFVSSNWAETPRLGKKNESSYFPGNVLTSTAVKDQNKHTASI NDLERETQPSYDIDNFDIDDFDDDDDWEDIMHNLAASKSSTAAYQPIKEGRPIKSVSERL SSAKTDCLPVSSTAQNINFSESIQNYTDKSAQNLASRNLKHERFQSLSFPHTKEMMKIFH KKFGLHNFRTNQLEAINAALLGEDCFILMPTGGGKSLCYQLPACVSPGVTVVISPLRSLI VDQVQKLTSLDIPATYLTGDKTDSEATNIYLQLSKKDPIIKLLYVTPEKICASNRLISTL ENLYERKLLARFVIDEAHCVSQWGHDFRQDYKRMNMLRQKFPSVPVMALTATANPRVQKD ILTQLKILRPQVFSMSFNRHNLKYYVLPKKPKKVAFDCLEWIRKHHPYDSGITYCLSRRE CDTMADTLQRDGLAALAYHAGLSDSARDEVQQKWINQDGCQVICATIAFGMGIDKPDVRF VIHASLPKSVEGYYQESGRAGRDGEISHCLLFYTYHDVTRLKRLIMMEKDGNHHTRETHF NNLYSMVHYCENITECRRIQLLAYFGENGFNPDFCKKHPDVSCDNCCKTKDYKTRDVTDD VKSIVRFVQEHSSSQGMRNIKHVGPSGRFTMNMLVDIFLGSKSAKIQSGIFGKGSAYSRH NAERLFKKLILDKILDEDLYINANDQATAYVMLGNKAQTVLNGNLKVDFMETENSSSVKK QKALVAKVSQREEMVKKCLGELTEVCKSLGKVFGVHYFNIFNTVTLKKLAESLSSDPEVL LQIDGVTEDKLEKYGAEVISVLQKYSEWTSPAEDSSPGISLSSSRGPGRSAAEELDEEIP VSSHYFASKTRNERKRKKMPASQRSKRRKTASSGSKAKGGSATCRKISSKTKSSSIIGSS SASHTSQATSGANSKLGIMAPPKPINRPFLKPSYAFS (BLM Isoform 1 CCDS 10363.1) (SEQ ID NO: 174) MAAVPQNNLQEQLERHSARTLNNKLSLSKPKFSGFIFKKKISSDNNVSVINVSVAKTPVL RNKDVNVIEDFSFSEPLPNTINQQRVKDFFKNAPAGQETQRGGSKSLLPDFLQTPKEVVC TTQNTPTVKKSRDTALKKLEFSSSPDSLSTINDWDDMDDFDTSETSKSFVTPPQSHFVRV STAQKSKKGKRNFFKAQLYTTNTVKTDLPPPSSESEQIDLTEEQKDDSEWLSSDVICIDD GPIAEVHINEDAQESDSLKTHLEDERDNSEKKKNLEEAELHSTEKVPCIEFDDDDYDTDF VPPSPEEIISASSSSSKCLSTLKDLDTSDRKEDVLSTSKDLLSKPEKMSMQELNPETSTD CDARQISLQQQLIHVMEHICKLIDTIPDDKLKLLDCGNELLQQRNIRRKLLTEVDFNKSD ASLLGSLWRYRPDSLDGPMEGDSCPTGNSMKELNFSHLPSNSVSPGDCLLTTTLGKTGFS ATRKNLFERPLFNTHLQKSFVSSNWAETPRLGKKNESSYFPGNVLTSTAVKDQNKHTASI NDLERETQPSYDIDNFDIDDFDDDDDWEDIMHNLAASKSSTAAYQPIKEGRPIKSVSERL SSAKTDCLPVSSTAQNINFSESIQNYTDKSAQNLASRNLKHERFQSLSFPHTKEMMKIFH KKFGLHNFRTNQLEAINAALLGEDCFILMPTGGGKSLCYQLPACVSPGVTVVISPLRSLI VDQVQKLTSLDIPATYLTGDKTDSEATNIYLQLSKKDPIIKLLYVTPEKICASNRLISTL ENLYERKLLARFVIDEAHCVSQWGHDFRQDYKRMNMLRQKFPSVPVMALTATANPRVQKD ILTQLKILRPQVFSMSFNRHNLKYYVLPKKPKKVAFDCLEWIRKHHPYDSGITYCLSRRE CDTMADTLQRDGLAALAYHAGLSDSARDEVQQKWINQDGCQVICATIAFGMGIDKPDVRF VIHASLPKSVEGYYQESGRAGRDGEISHCLLFYTYHDVTRLKRLIMMEKDGNHHTRETHF NNLYSMVHYCENITECRRIQLLAYFGENGFNPDFCKKHPDVSCDNCCKTKDYKTRDVTDD VKSIVRFVQEHSSSQGMRNIKHVGPSGRFTMNMLVDIFLESLSSDPEVLLQIDGVTEDKL EKYGAEVISVLQKYSEWTSPAEDSSPGISLSSSRGPGRSAAEELDEEIPVSSHYFASKTR NERKRKKMPASQRSKRRKTASSGSKAKGGSATCRKISSKTKSSSIIGSSSASHTSQATSG ANSKLGIMAPPKPINRPFLKPSYAFS (BLM Isoform 2 CCDS 73782.1) (SEQ ID NO: 175) RECQ1 (also called MASVSALTEELDSITSELHAVEIQIQELTERQQELIQKKKVLTKKIKQCLEDSDAGASNE RECQL1) YDSSPAAWNKEDFPWSGKVKDILQNVFKLEKFRPLQLETINVTMAGKEVFLVMPTGGGKS LCYQLPALCSDGFTLVICPLISLMEDQLMVLKQLGISATMLNASSSKEHVKWVHAEMVNK NSELKLIYVTPEKIAKSKMFMSRLEKAYEARRFTRIAVDEVHCCSQWGHDFRPDYKALGI LKRQFPNASLIGLTATATNHVLTDAQKILCIEKCFTFTASFNRPNLYYEVRQKPSNTEDF IEDIVKLINGRYKGQSGITYCFSQKDSEQVTVSLQNLGIHAGAYHANLEPEDKTTVHRKW SANEIQVVVATVAFGMGIDKPDVRFVIHHSMSKSMENYYQESGRAGRDDMKADCILYYGF GDIFRISSMVVMENVGQQKLYEMVSYCQNISKCRRVLMAQHFDEVWNSEACNKMCDNCCK DSAFERKNITEYCRDLIKILKQAEELNEKLTPLKLIDSWMGKGAAKLRVAGVVAPTLPRE DLEKIIAHFLIQQYLKEDYSFTAYATISYLKIGPKANLLNNEAHAITMQVTKSTQNSFRA ESSQTCHSEQGDKKMEEKNSGNFQKKAANMLQQSGSKNTGAKKRKIDDA (RECQL1 CCDS 31756.1) (SEQ ID NO: 176) WRN MSEKKLETTAQQRKCPEWMNVQNKRCAVEERKACVRKSVFEDDLPFLEFTGSIVYSYDAS DCSFLSEDISMSLSDGDVVGFDMEWPPLYNRGKLGKVALIQLCVSESKCYLFHVSSMSVF PQGLKMLLENKAVKKAGVGIEGDQWKLLRDFDIKLKNFVELTDVANKKLKCTETWSLNSL VKHLLGKQLLKDKSIRCSNWSKFPLTEDQKLYAATDAYAGFITYRNLEILDDTVQRFAIN KEEEILLSDMNKQLTSISEEVMDLAKHLPHAFSKLENPRRVSILLKDISENLYSLRRMII GSTNIETELRPSNNLNLLSFEDSTTGGVQQKQIREHEVLIHVEDETWDPTLDHLAKHDGE DVLGNKVERKEDGFEDGVEDNKLKENMERACLMSLDITEHELQILEQQSQEEYLSDIAYK STEHLSPNDNENDTSYVIESDEDLEMEMLKHLSPNDNENDTSYVIESDEDLEMEMLKSLE NLNSGTVEPTHSKCLKMERNLGLPTKEEEEDDENEANEGEEDDDKDFLWPAPNEEQVTCL KMYFGHSSFKPVQWKVIHSVLEERRDNVAVMATGYGKSLCFQYPPVYVGKIGLVISPLIS LMEDQVLQLKMSNIPACFLGSAQSENVLTDIKLGKYRIVYVTPEYCSGNMGLLQQLEADI GITLIAVDEAHCISEWGHDFRDSFRKLGSLKTALPMVPIVALTATASSSIREDIVRCLNL RNPQITCTGFDRPNLYLEVRRKTGNILQDLQPFLVKTSSHWEFEGPTIIYCPSRKMTQQV TGELRKLNLSCGTYHAGMSFSTRKDIHHRFVRDEIQCVIATIAFGMGINKADIRQVIHYG APKDMESYYQEIGRAGRDGLQSSCHVLWAPADINLNRHLLTEIRNEKFRLYKLKMMAKME KYLHSSRCRRQIILSHFEDKQVQKASLGIMGTEKCCDNCRSRLDHCYSMDDSEDTSWDFG PQAFKLLSAVDILGEKFGIGLPILFLRGSNSQRLADQYRRHSLFGTGKDQTESWWKAFSR QLITEGFLVEVSRYNKFMKICALTKKGRNWLHKANTESQSLILQANEELCPKKLLLPSSK TVSSGTKEHCYNQVPVELSTEKKSNLEKLYSYKPCDKISSGSNISKKSIMVQSPEKAYSS SQPVISAQEQETQIVLYGKLVEARQKHANKMDVPPAILATNKILVDMAKMRPTTVENVKR IDGVSEGKAAMLAPLLEVIKHFCQTNSVQTDLFSSTKPQEEQKTSLVAKNKICTLSQSMA ITYSLFQEKKMPLKSIAESRILPLMTIGMHLSQAVKAGCPLDLERAGLTPEVQKIIADVI RNPPVNSDMSKISLIRMLVPENIDTYLIHMAIEILKHGPDSGLQPSCDVNKRRCFPGSEE ICSSSKRSKEEVGINTETSSAERKRRLPVWFAKGSDTSKKLMDKTKRGGLFS (WRN CCDS 6082.1) (SEQ ID NO: 177) RTS (also called MERLRDVRERLQAWERAFRRQRGRRPSQDDVEAAPEETRALYREYRTLKRTTGQAGGGLRSSESLPAAAE RECQ4) EAPEPRCWGPHLNRAATKSPQSTPGRSRQGSVPDYGQRLKANLKGTLQAGPALGRRPWPLGRASSKASTP KPPGTGPVPSFAEKVSDEPPQLPEPQPRPGRLQHLQASLSQRLGSLDPGWLQRCHSEVPDFLGAPKACRP DLGSEESQLLIPGESAVLGPGAGSQGPEASAFQEVSIRVGSPQPSSSGGEKRRWNEEPWESPAQVQQESS QAGPPSEGAGAVAVEEDPPGEPVQAQPPQPCSSPSNPRYHGLSPSSQARAGKAEGTAPLHIFPRLARHDR GNYVRLNMKQKHYVRGRALRSRLLRKQAWKQKWRKKGECFGGGGATVTTKESCFLNEQFDHWAAQCPRPA SEEDTDAVGPEPLVPSPQPVPEVPSLDPTVLPLYSLGPSGQLAETPAEVFQALEQLGHQAFRPGQERAVM RILSGISTLLVLPTGAGKSLCYQLPALLYSRRSPCLTLVVSPLLSLMDDQVSGLPPCLKAACIHSGMTRK QRESVLQKIRAAQVHVLMLTPEALVGAGGLPPAAQLPPVAFACIDEAHCLSQWSHNFRPCYLRVCKVLRE RMGVHCFLGLTATATRRTASDVAQHLAVAEEPDLHGPAPVPTNLHLSVSMDRDTDQALLTLLQGKRFQNL DSIIIYCNRREDTERIAALLRTCLHAAWVPGSGGRAPKTTAEAYHAGMCSRERRRVQRAFMQGQLRVVVA TVAFGMGLDRPDVRAVLHLGLPPSFESYVQAVGRAGRDGQPAHCHLFLQPQGEDLRELRRHVHADSTDFL AVKRLVQRVFPACTCTCTRPPSEQEGAVGGERPVPKYPPQEAEQLSHQAAPGPRRVCMGHERALPIQLTV QALDMPEEAIETLLCYLELHPHHWLELLATTYTHCRLNCPGGPAQLQALAHRCPPLAVCLAQQLPEDPGQ GSSSVEFDMVKLVDSMGWELASVRRALCQLQWDHEPRTGVRRGTGVLVEFSELAFHLRSPGDLTAEEKDQ ICDFLYGRVQARERQALARLRRTFQAFHSVAFPSCGPCLEQQDEERSTRLKDLLGRYFEEEEGQEPGGME DAQGPEPGQARLQDWEDQVRCDIRQFLSLRPEEKFSSRAVARIFHGIGSPCYPAQVYGQDRRFWRKYLHL SFHALVGLATEELLQVAR (RECQ4, CCDS 75804.1) (SEQ ID NO: 178) RECQ5 MSSHHTTFPFDPERRVRSTLKKVFGFDSFKTPLQESATMAVVKGNKDVFVCMPTGAGKSLCYQLPALLAK GITIVVSPLIALIQDQVDHLLTLKVRVSSLNSKLSAQERKELLADLEREKPQTKILYITPEMAASSSFQP TLNSLVSRHLLSYLVVDEAHCVSQWGHDFRPDYLRLGALRSRLGHAPCVALTATATPQVQEDVFAALHLK KPVAIFKTPCFRANLFYDVQFKELISDPYGNLKDFCLKALGQEADKGLSGCGIVYCRTREACEQLAIELS CRGVNAKAYHAGLKASERTLVQNDWMEEKVPVIVATISFGMGVDKANVRFVAHWNIAKSMAGYYQESGRA GRDGKPSWCRLYYSRNDRDQVSFLIRKEVAKLQEKRGNKASDKATIMAFDALVTFCEELGCRHAAIAKYF GDALPACAKGCDHCQNPTAVRRRLEALERSSSWSKTCIGPSQGNGFDPELYEGGRKGYGDFSRYDEGSGG SGDEGRDEAHKREWNLFYQKQMQLRKGKDPKIEEFVPPDENCPLKEASSRRIPRLTVKAREHCLRLLEEA LSSNRQSTRTADEADLRAKAVELEHETFRNAKVANLYKASVLKKVADIHRASKDGQPYDMGGSAKSCSAQ AEPPEPNEYDIPPASHVYSLKPKRVGAGFPKGSCPFQTATELMETTRIREQAPQPERGGEHEPPSRPCGL LDEDGSEPLPGPRGEVPGGSAHYGGPSPEKKAKSSSGGSSLAKGRASKKQQLLATAAHKDSQSIARFFCR RVESPALLASAPEAEGACPSCEGVQGPPMAPEKYTGEEDGAGGHSPAPPQTEECLRERPSTCPPRDQGTP EVQPTPAKDTWKGKRPRSQQENPESQPQKRPRPSAKPSVVAEVKGSVSASEQGTLNPTAQDPFQLSAPGV SLKEAANVVVKCLTPFYKEGKFASKELFKGFARHLSHLLTQKTSPGRSVKEEAQNLIRHFFHGRARCESE ADWHGLCGPQR (ATP-dependent DNA helicase Q5 isoform 1 [Homo sapiens], CCDS 42380.1) (SEQ ID NO: 179) MSSHHTTFPFDPERRVRSTLKKVFGFDSFKTPLQESATMAVVKGNKDVFVCMPTGAGKSLCYQLPALLAK GITIVVSPLIALIQDQVDHLLTLKVRVSSLNSKLSAQERKELLADLEREKPQTKILYITPEMAASSSFQP TLNSLVSRHLLSYLVVDEAHCVSQWGHDFRPDYLRLGALRSRLGHAPCVALTATATPQVQEDVFAALHLK KPVAIFKTPCFRANLFYDVQFKELISDPYGNLKDFCLKALGQEADKGLSGCGIVYCRTREACEQLAIELS CRGVNAKAYHAGLKASERTLVQNDWMEEKVPVIVATISFGMGVDKANVRFVAHWNIAKSMAGYYQESGRA GRDGKPSWCRLYYSRNDRDQVSFLIRKEVAKLQEKRGNKASDKATIMAFDALVTFCEELGRWGRGHGKSL RAAWCSQVVSRHAEL (ATP-dependent DNA helicase Q5 isoform 2 [Homo sapiens], CCDS 32735.1) (SEQ ID NO: 180) MSSHHTTFPFDPERRVRSTLKKVFGFDSFKTPLQESATMAVVKGNKDVFVCMPTGAGKSLCYQLPALLAK GITIVVSPLIALIQDQVDHLLTLKVRVSSLNSKLSAQERKELLADLEREKPQTKILYITPEMAASSSFQP TLNSLVSRHLLSYLVVDEAHCVSQWGHDFRPDYLRLGALRSRLGHAPCVALTATATPQVQEDVFAALHLK KPVAIFKTPCFRANLFYDVQFKELISDPYGNLKDFCLKALGQEADKGLSGCGIVYCRTREACEQLAIELS CRGVNAKAYHAGLKASERTLVQNDWMEEKVPVIVATISFGMGVDKANVRFVAHWNIAKSMAGYYQESGRA GRDGKPSWCRLYYSRNDRDQVSFLIRKEVAKLQEKRGNKASDKATIMAFDALVTFCEELG (CCDS 45777.1) (SEQ ID NO: 181) RPA3 MVDMMDLPRSRINAGMLAQFIDKPVCFVGRLEKIHPTGKMFILSDGEGKNGTIELMEPLDEEISGIVEVV GRVTAKATILCTSYVQFKEDSHPFDLGLYNEAVKIIHDFPQFYPLGIVQHD (replication protein A3, 14 kDa, isoform CRA_a [Homo sapiens], CCDS 5356.1) (SEQ ID NO: 182) PP4 MAEISDLDRQIEQLRRCELIKESEVKALCAKAREILVEESNVQRVDSPVTVCGDIHGQFYDLKELFRVGG DVPETNYLFMGDFVDRGFYSVETFLLLLALKVRYPDRITLIRGNHESRQITQVYGFYDECLRKYGSVTVW RYCTEIFDYLSLSAIIDGKIFCVHGGLSPSIQTLDQIRTIDRKQEVPHDGPMCDLLWSDPEDTTGWGVSP RGAGYLFGSDVVAQFNAANDIDMICRAHQLVMEGYKWHFNETVLTVWSAPNYCYRCGNVAAILELDEHLQ KDFIIFEAAPQETRGIPSKKPVADYFL (PPP4C protein phosphatase 4, catalytic subunit, CCDS 10669.1) (SEQ ID NO: 183) RAD51 MAMQMQLEANADTSVEEESFGPQPISRLEQCGINANDVKKLEEAGFHTVEAVAYAPKKELINIKGISEAK ADKILAEAAKLVPMGFTTATEFHQRRSEIIQITTGSKELDKLLQGGIETGSITEMFGEFRTGKTQICHTL AVTCQLPIDRGGGEGKAMYIDTEGTFRPERLLAVAERYGLSGSDVLDNVAYARAFNTDHQTQLLYQASAM MVESRYALLIVDSATALYRTDYSGRGELSARQMHLARFLRMLLRLADEFGVAVVITNQVVAQVDGAAMFA ADPKKPIGGNITAHASTTRLYLRKGRGETRICKIYDSPCLPEAEAMFAINADGVGDAKD (RAD51 [Homo sapiens], CCDS 10062.1) (SEQ ID NO: 184) MAMQMQLEANADTSVEEESFGPQPISRLEQCGINANDVKKLEEAGFHTVEAVAYAPKKELINIKGISEAK ADKILTESRSVARLECNSVILVYCTLRLSGSSDSPASASRVVGTTGGIETGSITEMFGEFRTGKTQICHT LAVTCQLPIDRGGGEGKAMYIDTEGTFRPERLLAVAERYGLSGSDVLDNVAYARAFNTDHQTQLLYQASA MMVESRYALLIVDSATALYRTDYSGRGELSARQMHLARFLRMLLRLADEFGVAVVITNQVVAQVDGAAMF AADPKKPIGGNITAHASTTRLYLRKGRGETRICKIYDSPCLPEAEAMFAINADGVGDAKD (RAD51 [Homo sapiens], CCDS 53931.1) (SEQ ID NO: 185) MAMQMQLEANADTSVEEESFGPQPISRLEQCGINANDVKKLEEAGFHTVEAVAYAPKKELINIKGISEAK ADKILAEAAKLVPMGFTTATEFHQRRSEIIQITTGSKELDKLLQGGIETGSITEMFGEFRTGKTQICHTL AVTCQLPIDRGGGEGKAMYIDTEGTFRPERLLAVAERYGLSGSDVLDNVAYARAFNTDHQTQLLYQASAM MVESRYALLIVDSATALYRTDYSGRGELSARQMHLARFLRMLLRLADEIVSEERKRGNQNLQNLRLSLSS (CCDS 53932.1) (SEQ ID NO: 186) BACH1 MSLSENSVFAYESSVHSTNVLLSLNDQRKKDVLCDVTIFVEGQRFRAHRSVLAACSSYFHSRIVGQADGE LNITLPEEVTVKGFEPLIQFAYTAKLILSKENVDEVCKCVEFLSVHNIEESCFQFLKFKFLDSTADQQEC PRKKCFSSHCQKTDLKLSLLDQRDLETDEVEEFLENKNVQTPQCKLRRYQGNAKASPPLQDSASQTYESM CLEKDAALALPSLCPKYRKFQKAFGTDRVRTGESSVKDIHASVQPNERSENECLGGVPECRDLQVMLKCD ESKLAMEPEETKKDPASQCPTEKSEVTPFPHNSSIDPHGLYSLSLLHTYDQYGDLNFAGMQNTTVLTEKP LSGTDVQEKTFGESQDLPLKSDLGTREDSSVASSDRSSVEREVAEHLAKGFWSDICSTDTPCQMQLSPAV AKDGSEQISQKRSECPWLGIRISESPEPGQRTFTTLSSVNCPFISTLSTEGCSSNLEIGNDDYVSEPQQE PCPYACVISLGDDSETDTEGDSESCSAREQECEVKLPFNAQRIISLSRNDFQSLLKMHKLTPEQLDCIHD IRRRSKNRIAAQRCRKRKLDCIQNLESEIEKLQSEKESLLKERDHILSTLGETKQNLTGLCQKVCKEAAL SQEQIQILAKYSAADCPLSFLISEKDKSTPDGELALPSIFSLSDRPPAVLPPCARGNSEPGYARGQESQQ MSTATSEQAGPAEQCRQSGGISDFCQQMTDKCTTDE (transcription regulator protein BACH1 [Homo sapiens] CCDS 13585.1) (SEQ ID NO: 187) FANCJ MSSMWSEYTIGGVKIYFPYKAYPSQLAMMNSILRGLNSKQHCLLESPTGSGKSLALLCSA LAWQQSLSGKPADEGVSEKAEVQLSCCCACHSKDFTNNDMNQGTSRHFNYPSTPPSERNG TSSTCQDSPEKTTLAAKLSAKKQASIYRDENDDFQVEKKRIRPLETTQQIRKRHCFGTEV HNLDAKVDSGKTVKLNSPLEKINSFSPQKPPGHCSRCCCSTKQGNSQESSNTIKKDHTGK SKIPKIYFGTRTHKQIAQITRELRRTAYSGVPMTILSSRDHTCVHPEVVGNFNRNEKCME LLDGKNGKSCYFYHGVHKISDQHTLQTFQGMCKAWDIEELVSLGKKLKACPYYTARELIQ DADIIFCPYNYLLDAQIRESMDLNLKEQVVILDEAHNIEDCARESASYSVTEVQLRFARD ELDSMVNNNIRKKDHEPLRAVCCSLINWLEANAEYLVERDYESACKIWSGNEMLLTLHKM GITTATFPILQGHFSAVLQKEEKISPIYGKEEAREVPVISASTQIMLKGLFMVLDYLFRQ NSRFADDYKIAIQQTYSWTNQIDISDKNGLLVLPKNKKRSRQKTAVHVLNFWCLNPAVAF SDINGKVQTIVLTSGTLSPMKSFSSELGVTFTIQLEANHIIKNSQVWVGTIGSGPKGRNL CATFQNTETFEFQDEVGALLLSVCQTVSQGILCFLPSYKLLEKLKERWLSTGLWHNLELV KTVIVEPQGGEKTNFDELLQVYYDAIKYKGEKDGALLVAVCRGKVSEGLDFSDDNARAVI TIGIPFPNVKDLQVELKRQYNDHHSKLRGLLPGRQWYEIQAYRALNQALGRCIRHRNDWG ALILVDDRFRNNPSRYISGLSKWVRQQIQHHSTFESALESLAEFSKKHQKVLNVSIKDRT NIQDNESTLEVTSLKYSTSPYLLEAASHLSPENFVEDEAKICVQELQCPKIITKNSPLPS SIISRKEKNDPVFLEEAGKAEKIVISRSTSPTFNKQTKRVSWSSFNSLGQYFTGKIPKAT PELGSSENSASSPPRFKTEKMESKTVLPFTDKCESSNLTVNTSFGSCPQSETIISSLKID ATLTRKNHSEHPLCSEEALDPDIELSLVSEEDKQSTSNRDFETEAEDESIYFTPELYDPE DTDEEKNDLAETDRGNRLANNSDCILAKDLFEIRTIKEVDSAREVKAEDCIDTKLNGILH IEESKIDDIDGNVKTTWINELELGKTHEIEIKNFKPSPSKNKGMFPGFK (FancJ CCDS 11631.1) (SEQ ID NO: 188) Topbp1 MSRNDKEPFFVKFLKSSDNSKCFFKALESIKEFQSEEYLQIITEEEALKIKENDRSLYICDPFSGVVFDH LKKLGCRIVGPQVVIFCMHHQRCVPRAEHPVYNMVMSDVTISCTSLEKEKREEVHKYVQMMGGRVYRDLN VSVTHLIAGEVGSKKYLVAANLKKPILLPSWIKTLWEKSQEKKITRYTDINMEDFKCPIFLGCTICVTGL CGLDRKEVQQLTVKHGGQYMGQLKMNECTHLIVQEPKGQKYECAKRWNVHCVTTQWFFDSIEKGFCQDES IYKTEPRPEAKTMPNSSTPTSQINTIDSRTLSDVSNISNINASCVSESICNSLNSKLEPTLENLENLDVS AFQAPEDLLDGCRIYLCGFSGRKLDKLRRLINSGGGVRFNQLNEDVTHVIVGDYDDELKQFWNKSAHRPH VVGAKWLLECFSKGYMLSEEPYIHANYQPVEIPVSHKPESKAALLKKKNSSFSKKDFAPSEKHEQADEDL LSQYENGSSTVVEAKTSEARPFNDSTHAEPLNDSTHISLQEENQSSVSHCVPDVSTITEEGLFSQKSFLV LGFSNENESNIANIIKENAGKIMSLLSRTVADYAVVPLLGCEVEATVGEVVTNTWLVTCIDYQTLFDPKS NPLFTPVPVMTGMTPLEDCVISFSQCAGAEKESLTFLANLLGASVQEYFVRKSNAKKGMFASTHLILKER GGSKYEAAKKWNLPAVTIAWLLETARTGKRADESHFLIENSTKEERSLETEITNGINLNSDTAEHPGTRL QTHRKTVVTPLDMNRFQSKAFRAVVSQHARQVAASPAVGQPLQKEPSLHLDTPSKFLSKDKLFKPSFDVK DALAALETPGRPSQQKRKPSTPLSEVIVKNLQLALANSSRNAVALSASPQLKEAQSEKEEAPKPLHKVVV CVSKKLSKKQSELNGIAASLGADYRWSFDETVTHFIYQGRPNDTNREYKSVKERGVHIVSEHWLLDCAQE CKHLPESLYPHTYNPKMSLDISAVQDGRLCNSRLLSAVSSTKDDEPDPLILEENDVDNMATNNKESAPSN GSGKNDSKGVLTQTLEMRENFQKQLQEIMSATSIVKPQGQRTSLSRSGCNSASSTPDSTRSARSGRSRVL EALRQSRQTVPDVNTEPSQNEQIIWDDPTAREERARLASNLQWPSCPTQYSELQVDIQNLEDSPFQKPLH DSEIAKQAVCDPGNIRVTEAPKHPISEELETPIKDSHLIPTPQAPSIAFPLANPPVAPHPREKIITIEET HEELKKQYIFQLSSLNPQERIDYCHLIEKLGGLVIEKQCFDPTCTHIVVGHPLRNEKYLASVAAGKWVLH RSYLEACRTAGHFVQEEDYEWGSSSILDVLTGINVQQRRLALAAMRWRKKIQQRQESGIVEGAFSGWKVI LHVDQSREAGFKRLLQSGGAKVLPGHSVPLFKEATHLFSDLNKLKPDDSGVNIAEAAAQNVYCLRTEYIA DYLMQESPPHVENYCLPEAISFIQNNKELGTGLSQKRKAPTEKNKIKRPRVH (Topbp1, CCDS 46919.1) (SEQ ID NO: 189) TOPO III MIFPVARYALRWLRRPEDRAFSRAAMEMALRGVRKVLCVAEKNDAAKGIADLLSNGRMRRREGLSKFNKI YEFDYHLYGQNVTMVMTSVSGHLLAHDFQMQFRKWQSCNPLVLFEAEIEKYCPENFVDIKKTLERETRQC QALVIWTDCDREGENIGFEIIHVCKAVKPNLQVLRARFSEITPHAVRTACENLTEPDQRVSDAVDVRQEL DLRIGAAFTRFQTLRLQRIFPEVLAEQLISYGSCQFPTLGFVVERFKAIQAFVPEIFHRIKVTHDHKDGI VEFNWKRHRLFNHTACLVLYQLCVEDPMATVVEVRSKPKSKWRPQALDTVELEKLASRKLRINAKETMRI AEKLYTQGYISYPRTETNIFPRDLNLTVLVEQQTPDPRWGAFAQSILERGGPTPRNGNKSDQAHPPIHPT KYTNNLQGDEQRLYEFIVRHFLACCSQDAQGQETTVEIDIAQERFVAHGLMILARNYLDVYPYDHWSDKI LPVYEQGSHFQPSTVEMVDGETSPPKLLTEADLIALMEKHGIGTDATHAEHIETIKARMYVGLTPDKRFL PGHLGMGLVEGYDSMGYEMSKPDLRAELEADLKLICDGKKDKFVVLRQQVQKYKQVFIEAVAKAKKLDEA LAQYFGNGTELAQQEDIYPAMPEPIRKCPQCNKDMVLKTKKNGGFYLSCMGFPECRSAVWLPDSVLEASR DSSVCPVCQPHPVYRLKLKFKRGSLPPTMPLEFVCCIGGCDDTLREILDLRFSGGPPRASQPSGRLQANQ SLNRMDNSQHPQPADSRQTGSSKALAQTLPPPTAAGESNSVTCNCGQEAVLLTVRKEGPNRGRQFFKCNG GSCNFFLWADSPNPGAGGPPALAYRPLGASLGCPPGPGIHLGGFGNPGDGSGSGTSCLCSQPSVTRTVQK DGPNKGRQFHTCAKPREQQCGFFQWVDENTAPGTSGAPSWTGDRGRTLESEARSKRPRASSSDMGSTAKK PRKCSLCHQPGHTRPFCPQNR (DNA topoisomerase III [Homo sapiens], CCDS 11194.1) (SEQ ID NO: 190) FEN1 MGIQGLAKLIADVAPSAIRENDIKSYFGRKVAIDASMSIYQFLIAVRQGGDVLQNEEGET TSHLMGMFYRTIRMMENGIKPVYVFDGKPPQLKSGELAKRSERRAEAEKQLQQAQAAGAE QEVEKFTKRLVKVTKQHNDECKHLLSLMGIPYLDAPSEAEASCAALVKAGKVYAAATEDM DCLTFGSPVLMRHLTASEAKKLPIQEFHLSRILQELGLNQEQFVDLCILLGSDYCESIRG IGPKRAVDLIQKHKSIEEIVRRLDPNKYPVPENWLHKEAHQLFLEPEVLDPESVELKWSE PNEEELIKFMCGEKQFSEERIRSGVKRLSKSRQGSTQGRLDDFFKVTGSLSSAKRKEPEP KGSTKKKAKTGAAGKFKRGK (Fen1 CCDS 8010.1) (SEQ ID NO: 191) MUS81 MAAPVRLGRKRPLPACPNPLFVRWLTEWRDEATRSRRRTRFVFQKALRSLRRYPLPLRSGKEAKILQHFG DGLCRMLDERLQRHRTSGGDHAPDSPSGENSPAPQGRLAEVQDSSMPVPAQPKAGGSGSYWPARHSGARV ILLVLYREHLNPNGHHFLTKEELLQRCAQKSPRVAPGSARPWPALRSLLHRNLVLRTHQPARYSLTPEGL ELAQKLAESEGLSLLNVGIGPKEPPGEETAVPGAASAELASEAGVQQQPLELRPGEYRVLLCVDIGETRG GGHRPELLRELQRLHVTHTVRKLHVGDFVWVAQETNPRDPANPGELVLDHIVERKRLDDLCSSIIDGRFR EQKFRLKRCGLERRVYLVEEHGSVHNLSLPESTLLQAVTNTQVIDGFFVKRTADIKESAAYLALLTRGLQ RLYQGHTLRSRPWGTPGNPESGAMTSPNPLCSLLTFSDFNAGAIKNKAQSVREVFARQLMQVRGVSGEKA AALVDRYSTPASLLAAYDACATPKEQETLLSTIKCGRLQRNLGPALSRTLSQLYCSYGPLT (MUS81 endonuclease homolog (yeast), isoform CRA_b [Homo sapiens], CCDS 8115.1) (SEQ ID NO: 192) EME1 MALKKSSPSLDSGDSDSEELPTFAFLKKEPSSTKRRQPEREEKIVVVDISDCEASCPPAPELFSPPVPEI AETVTQTQPVRLLSSESEDEEEFIPLAQRLTCKFLTHKQLSPEDSSSPVKSVLDHQNNEGASCDWKKPFP KIPEVPLHDTPERSAADNKDLILDPCCQLPAYLSTCPGQSSSLAVTKTNSDILPPQKKTKPSQKVQGRGS HGCRQQRQARQKESTLRRQERKNAALVTRMKAQRPEECLKHIIVVLDPVLLQMEGGGQLLGALQTMECRC VIEAQAVPCSVTWRRRAGPSEDREDWVEEPTVLVLLRAEAFVSMIDNGKQGSLDSTMKGKETLQGFVTDI TAKTAGKALSLVIVDQEKCFSLELLFFDFLPCTSAQNPPRRGKQGANKQTKKQQQRQPEASIGSMVSRVD AEEALVDLQLHTEAQAQIVQSWKELADFTCAFTKAVAEAPFKKLRDETTFSFCLESDWAGGVKVDLAGRG LALVWRRQIQQLNRVSLEMASAVVNAYPSPQLLVQAYQQCFSDKERQNLLADIQVRRGEGVTSTSRRIGP ELSRRIYLQMTTLQPHLSLDSAD (crossover junction endonuclease EME1 isoform 2 [Homo sapiens], CCDS 54141.1) (SEQ ID NO: 193) MALKKSSPSLDSGDSDSEELPTFAFLKKEPSSTKRRQPEREEKIVVVDISDCEASCPPAPELFSPPVPEI AETVTQTQPVRLLSSESEDEEEFIPLAQRLTCKFLTHKQLSPEDSSSPVKSVLDHQNNEGASCDWKKPFP KIPEVPLHDTPERSAADNKDLILDPCCQLPAYLSTCPGQSSSLAVTKTNSDILPPQKKTKPSQKVQGRGS HGCRQQRQARQKESTLRRQERKNAALVTRMKAQRPEECLKHIIVVLDPVLLQMEGGGQLLGALQTMECRC VIEAQAVPCSVTWRRRAGPSEDREDWVEEPTVLVLLRAEAFVSMIDNGKQGSLDSTMKGKETLQGFVTDI TAKTAGKALSLVIVDQEKCFSAQNPPRRGKQGANKQTKKQQQRQPEASIGSMVSRVDAEEALVDLQLHTE AQAQIVQSWKELADFTCAFTKAVAEAPFKKLRDETTFSFCLESDWAGGVKVDLAGRGLALVWRRQIQQLN RVSLEMASAVVNAYPSPQLLVQAYQQCFSDKERQNLLADIQVRRGEGVTSTSRRIGPELSRRIYLQMTTL QPHLSLDSAD (crossover junction endonuclease EME1 isoform 1 [Homo sapiens], CCDS 11565.1) (SEQ ID NO: 194) SLX1 MGPAGVAARPGRFFGVYLLYCLNPRYRGRVYVGFIVNTARRVQQHNGGRKKGGAWRISGRGPWEMVLVVH GFPSSVAALRFEWAWQHPHASRRLAHVGPRLRGETAFAFHLRVLAHMLRAPPWARLPLTLRWVRPDLRQD LCLPPPPHVPLAFGPPPPQAPAPRRRAGPFDDAEPEPDQGDPGACCSLCAQTIQDEEGPLCCPHPGCLLR AHVICLAEEFLQEEPGQLLPLEGQCPCCEKSLLWGDLIWLCQMDTEKEVEDSELEEAHWTDLLET (structure-specific endonuclease subunit SLX1 isoform 2 [Homo sapiens], CCDS 32431.1) (SEQ ID NO: 195) MGPAGVAARPGRFFGVYLLYCLNPRYRGRVYVGFIVNTARRVQQHNGGRKKGGAWRISGRGPWEMVLVVH GFPSSVAALRDEEGPLCCPHPGCLLRAHVICLAEEFLQEEPGQLLPLEGQCPCCEKSLLWGDLIWLCQMD TEKEVEDSELEEAHWTDLLET (structure-specific endonuclease subunit SLX1 isoform 1 [Homo sapiens], CCDS 32432.1) (SEQ ID NO: 196) SLX4 MKLSVNEAQLGFYLGSLSHLSACPGIDPRSSEDQPESLKTGQMMDESDEDFKELCASFFQRVKKHGIKEV SGERKTQKAASNGTQIRSKLKRTKQTATKTKTLQGPAEKKPPSGSQAPRTKKQRVTKWQASEPAHSVNGE GGVLASAPDPPVLRETAQNTQTGNQQEPSPNLSREKTRENVPNSDSQPPPSCLTTAVPSPSKPRTAQLVL QRMQQFKRADPERLRHASEECSLEAAREENVPKDPQEEMMAGNVYGLGPPAPESDAAVALTLQQEFARVG ASAHDDSLEEKGLFFCQICQKNLSAMNVTRREQHVNRCLDEAEKTLRPSVPQIPECPICGKPFLTLKSRT SHLKQCAVKMEVGPQLLLQAVRLQTAQPEGSSSPPMFSFSDHSRGLKRRGPTSKKEPRKRRKVDEAPSED LLVAMALSRSEMEPGAAVPALRLESAFSERIRPEAENKSRKKKPPVSPPLLLVQDSETTGRQIEDRVALL LSEEVELSSTPPLPASRILKEGWERAGQCPPPPERKQSFLWEGSALTGAWAMEDFYTARLVPPLVPQRPA QGLMQEPVPPLVPPEHSELSERRSPALHGTPTAGCGSRGPSPSASQREHQALQDLVDLAREGLSASPWPG SGGLAGSEGTAGLDVVPGGLPLIGFVVPSQDKHPDRGGRILLSLGLLVADFGAMVNNPHLSDVQFQTDSG EVLYAHKFVLYARCPLLIQYVNNEGFSAVEDGVLTQRVLLGDVSTEAARTFLHYLYTADTGLPPGLSSEL SSLAHRFGVSELVHLCEQVPIATDSEGKPWEEKEAENCESRAENFQELLRSMWADEEEEAETLLKSKDHE EDQENVNEAEMEEIYEFAATQRKLLQEERAAGAGEDADWLEGGSPVSGQLLAGVQVQKQWDKVEEMEPLE PGRDEAATTWEKMGQCALPPPQGQHSGARGAEAPEQEAPEEALGHSSCSSPSRDCQAERKEGSLPHSDDA GDYEQLFSSTQGEISEPSQITSEPEEQSGAVRERGLEVSHRLAPWQASPPHPCRFLLGPPQGGSPRGSHH TSGSSLSTPRSRGGTSQVGSPTLLSPAVPSKQKRDRSILTLSKEPGHQKGKERRSVLECRNKGVLMFPEK SPSIDLTQSNPDHSSSRSQKSSSKLNEEDEVILLLDSDEELELEQTKMKSISSDPLEEKKALEISPRSCE LFSIIDVDADQEPSQSPPRSEAVLQQEDEGALPENRGSLGRRGAPWLFCDRESSPSEASTTDTSWLVPAT PLASRSRDCSSQTQISSLRSGLAVQAVTQHTPRASVGNREGNEVAQKFSVIRPQTPPPQTPSSCLTPVSP GTSDGRRQGHRSPSRPHPGGHPHSSPLAPHPISGDRAHFSRRFLKHSPPGPSFLNQTPAGEVVEVGDSDD EQEVASHQANRSPPLDSDPPIPIDDCCWHMEPLSPIPIDHWNLERTGPLSTSSPSRRMNEAADSRDCRSP GLLDTTPIRGSCTTQRKLQEKSSGAGSLGNSRPSFLNSALWDVWDGEEQRPPETPPPAQMPSAGGAQKPE GLETPKGANRKKNLPPKVPITPMPQYSIMETPVLKKELDRFGVRPLPKRQMVLKLKEIFQYTHQTLDSDS EDESQSSQPLLQAPHCQTLASQTYKPSRAGVHAQQEATTGPGAHRPKGPAKTKGPRHQRKHHESITPPSR SPTKEAPPGLNDDAQIPASQESVATSVDGSDSSLSSQSSSSCEFGAAFESAGEEEGEGEVSASQAAVQAA DTDEALRCYIRSKPALYQKVLLYQPFELRELQAELRQNGLRVSSRRLLDFLDTHCITFTTAATRREKLQG RRRQPRGKKKVERN (structure-specific endonuclease subunit SLX4 [Homo sapiens], CCDS 10506.2) (SEQ ID NO: 197) a FA protein —

In some embodiments, the methods described herein involve down-regulating one HDR pathway in order to promote another HDR pathway. For example, the SSA pathway may be down-regulated in order to promote HR and/or alt-HR. In another embodiment, the alt-HR pathway may be downregulate in order to promote HR and/or SSA. In another embodiment, the alt-HR pathway may be downregulated to promote SSA and HR.

For instance, the methods may involve modulating, e.g., inhibiting, a component (e.g., exactly one component, or one or more components, e.g., two or three components) of the HDR pathway, e.g., a component of Table VI.1(C) or VI.2. More specifically, the methods may involve modulating, e.g., inhibiting, exactly one of, or one or more of a PARP, PARP1, PARP2, MRN complex, MRE11, RAD50, NBS1, ATM, BRCA2, BRCA1, BRCA1 complex, BRCA1-C complex, CtIP, EX01, BLM, PP4, RAD51, FEN1, MUS81/EME1, SLX1/SLX4, and a FA protein. In some embodiments, one or more of alt-HR, anti-HR, NHEJ, MMEJ, SSA, SSBR, MMR, NER, and BER, are not substantially down-regulated, e.g., in some embodiments the only DNA damage repair pathway to be substantially down-regulated is one HDR pathway. In some embodiments, a PARP (e.g., a PARP which is involved in NHEJ and/or SSBr) is not down-regulated. In some embodiments, XRCC1 (e.g., involved in NER, alt-NHEJ, BER, and/or SSBr) is not down-regulated.

In some embodiments, RTEL is inhibited in order to promote HDR.

In some embodiments, an HDR pathway is down-regulated using an siRNA against a component of the pathway, e.g., BRCA2, BRCA1, or Rad51, or a Rad51 inhibitor such as B02, A03, AI-10, RI-1, RI-2, or IBR2, or an agent of Table VI.3, or any combination thereof. Down-regulation may result in enhanced alt-HR repair (see below). In some embodiments, an HDR pathway is down regulated using an HDR-enhancing gRNA that targets a component of the HDR pathway, e.g., BRCA2, BRCA1, or Rad51. In some embodiments, the HDR-enhancing gRNA is used in combination with an eiCas9 molecule. In some embodiments, the HDR-enhancing gRNA is used in combination with an eaCas9 molecule. In some embodiments, the HDR-enhancing gRNA is administered to the cell as a HDR-enhancing gRNA:Cas9 molecule complex.

TABLE VI.3 Down-regulators of HDR. (In this table and throughout this disclosure, when siRNAs are written with both upper- case and lower-case letters, the upper-case letters indicate ribonucleotides and the lower-case letters indicate deoxyribonucleotides.) 1. BRCA1 siRNA BRCA1 siRNA (h), sold by Santa Cruz Biotechnology; BRCA1 siRNA II, sold by Cell Signaling Technology Entrez Entrez Gene Gene GenBank Reagent SEQ Symbol ID Acc. No. Source ID ID Sequence (sense) BRCA1 672 NM_007294 D-003461-05 198 CAGCUACCCUUCCAUCAUA BRCA1 672 NM_007294 D-003461-06 199 GGGAUACCAUGCAACAUAA BRCA1 672 NM_007294 D-003461-07 200 GAAGGAGCUUUCAUCAUUC BRCA1 672 NM_007294 D-003461-08 203 CUAGAAAUCUGUUGCUAUG BRCA1 672 NM_007294 s224683 204 UUCUUUAAUAGACUGGGUCac BRCA1 672 NM_007294 s458 205 UAUGAUGGAAGGGUAGCUGtt BRCA1 672 NM_007294 s459 206 UAUCAGGUUAUGUUGCAUGgt antibodies BRCA1 (D-9) antibody, sold by Santa Cruz Biotechnology; BRCA1 (D-20) antibody, sold by Santa Cruz Biotechnology; BRCA1 (GLK-2) antibody, sold by Santa Cruz Biotechnology; BRCA1 (C-20) antibody, sold by Santa Cruz Biotechnology; BRCA1 (287.17) antibody, sold by Santa Cruz Biotechnology; BRCA1 antibody, sold by Cell Signaling Technology 2. BRCA2 siRNA BRCA2 silencer # 121226, sold by Life Technologies; BRCA2-set siRNA/shRNA/RNAi Lentivector, sold by ABM Entrez Entrez Gene Gene GenBank Reagent SEQ Symbol ID Acc. No. Source ID ID Sequence (sense) BRCA2 675 NM_000059 D-003462-01 207 GAAACGGACUUGCUAUUUA BRCA2 675 NM_000059 D-003462-02 208 GUAAAGAAAUGCAGAAUUC BRCA2 675 NM_000059 D-003462-03 209 GGUAUCAGAUGCUUCAUUA BRCA2 675 NM_000059 D-003462-04 210 GAAGAAUGCAGGUUUAAUA BRCA2 675 NM_000059 s2083 211 UUCCGUUUAAUUUCAACUGta BRCA2 675 NM_000059 s2084 212 UUGCGAAAUAUGUAUAAUCca BRCA2 675 NM_000059 s224695 213 UACGUUUUUAGGUGAAGCCtg antibodies Anti-BRCA2 antibody (ab27976), sold by Abcam; Anti-BRCA2 antibody (ab9143), sold by Abcam; Anti-BRCA2 antibody (ab123491), sold by Abcam 3. Rad51 siRNA Rad51 siRNA (m), sold by Santa Cruz Biotechnology siRNA from siRNA plasmid, pKD-Rad51-v1, sold by EMD Millipore Entrez Entrez Gene Gene GenBank Reagent SEQ Symbol ID Acc. No. Source ID ID Sequence (sense) RAD51 5888 NM_002875 D-003530-05 214 GCAGUGAUGUCCUGGAUAA RAD51 5888 NM_002875 D-003530-07 215 CCAACGAUGUGAAGAAAUU RAD51 5888 NM_002875 D-003530-08 216 AAGCUAUGUUCGCCAUUAA RAD51 5888 NM_002875 s11734 217 UGCAUACCUAGAUUCUACCat RAD51 5888 NM_002875 s11735 218 UGAUUAGUGAUUACCACUGct RAD51 5888 NM_002875 s11736 219 UGCUUGAUAAAGGAGCUGGgt Antibodies RAD51 mouse monoclonal antibody (clone 3C10), sold by Life Technologies; Rad51 (3C10) antibody, sold by Santa Cruz Biotechnology; Rad51 (F-11) antibody, sold by Santa Cruz Biotechnology; Rad51 (G-9) antibody, sold by Santa Cruz Biotechnology; Compounds B02 Huang et al. (2011) ACS CHEM. BIOL. 6(6): 628-35. A03 Huang et al. (2011) ACS CHEM. BIOL. 6(6): 628-35. RI-1 Budke et al. (2012) NUCLEIC ACIDS RES. 40(15): 7347-57. IBR2 Zhu et al. (2013) EMBO MOL. MED. 5(3): 353-65.

HDR and the Cell Cycle

HDR is highly influenced by the stage of the cell cycle. Cell cycle regulation acts on several points of the pathway, which are discussed below.

First, in the wild-type context, HDR is thought to be limited to S and G2 phase because HDR requires RAD51 to interact with the C-terminus of BRCA2, and this interaction only occurs during S and G2 phases.

In addition, cell cycle-dependent differences in resection may help limit homologous recombination to S/G2. In mammals, to promote HDR, the resecting endonuclease CtIP is activated by CDK phosphorylation at threonine 847. A further phosphorylation on serine 327 promotes CtIP's interaction with the BRCT domain of BRCA1 (which is important for HDR). In a mutually antagonistic arrangement, BRCA1-CtIP favors homologous recombination by antagonizing 53BP1-RIF1 in G2, whereas in G1, 53BP1-RIF2 blocks BRCA1 from accumulating at DSBs. Resection involves not only nucleases but helicases. Helicases are motor proteins that move along the backbone of the DNA and alter the structure of DNA by unwinding DNA or promoting the annealing of single strands. Unwinding of the DNA occurs through an ATP-dependent process that breaks the hydrogen bonds between the nucleotides of annealed strands, e.g., through ATP hydrolysis. Unwinding activity can occur in the 5′ to 3′ direction or in the 3′ to 5′ direction. Helicase activity also includes promoting ATP-independent or ATP-dependent annealing of two single strands with significant or sufficient complementarity. The helicase BLM also undergoes cell-cycle dependent regulation. More particularly, sumoylation of BLM appears to promote recombination.

The stage of the cell cycle also affects the formation of the RAD51 filament. For instance, RPA (which forms a substrate for assembly of the RAD51 filament) is phosphorylated in a cell cycle dependent manner, affecting its ability to localize properly. In addition, RAD51 is more directly regulated; the CHK1 kinase phosphorylates it, allowing it to form foci at the sites of DNA damage and promote repair. As an additional form of regulation, CDK-cyclin A phosphorylates BRCA2 in M phase, preventing BRCA2 from interacting with RAD51, effectively shutting down HDR at the end of G2 phase.

In addition, the MRN complex may also be a target of cell cycle dependent regulation.

VI.2 Antirecombinant Factors

The anti-homologous recombination (anti-HR) pathway is an HDR pathway and involves helicases that disrupt RAD51 ssDNA filaments. In mammals, the helicase FBH1 is thought to prevent RAD51 from localizing to breaks (e.g., by replacing Rad51 on a filament), thereby suppressing HDR. FBH1's role may be to prevent spontaneous synthesis dependent strand annealing that initiates inappropriately. A second helicase, RECQ5, that has a 3′ to 5′ activity, can suppress HDR by binding RAD51 and displacing it from the ssDNA. A third helicase, BLM, can also bind RAD51 and disrupt RAD51 ssDNA filaments. BLM and RECQ5 are both members of the RecQ helicase family and act in the 3′ to 5′ direction. A fourth helicase, FANCJ, acts in the 5′ to 3′ direction and can disrupt RAD51 ssDNA filaments. A fifth anti-recombination helicase is WRN (Werner). A sixth protein, PARI, has a helicase domain but lacks Walker A and B motifs, so it may not be an active helicase. PARI can replace a Rad51 filament in vitro. Lastly, RTEL has been shown to displace Rad51. Regulator of telomere elongation helicase (RTEL or RTEL1) has ATP-dependent 5′ to 3′ DNA helicase activity.

Another class of anti-recombination factors disrupts D-loops. These factors include RECQl, BLM, and WRN.

Rap80 also acts against homologous recombination. The BRCA1-RAP80 complex restricts end resection in S/G(2) phase of the cell cycle, thereby limiting HDR. It recruits the BRCA1 A complex and that prevents the repair throughout HR (see Hu Y. et al. (2011) GENES DEV. 25(7): 685-700; and Coleman and Greenberg (2011) J. BIOL. CHEM. 286(15): 13669-80.

Several miRNAs are also involved in anti-HR: miR-545 (which downregulates BRCA1 and opposes Rad51 focus formation), miR-107 and miR-155 (which downregulate Rad51), miR-1255 (which downregulates BRCA1 and BRCA2), miR-148 (which downregulates Rad51, especially in G1), and miR-193 (which downregulates BRCA1, BRCA2, and Rad51). Anti-miRs can increase the levels of homologous repair factors in G1.

In one embodiment, HDR repair pathways can be promoted by down-regulating the antirecombinant factors of the anti-HR pathway. For example, in one embodiment, a Cas9 molecule and gRNA can induce a DSB in a desired location during G2 or another phase of the cycle. This DSB can be formed using, e.g., one Cas9 molecule with the ability to produce DSBs, or two nickases. A template nucleic acid can be added to the cell, so that the HDR machinery repairs the DSB using the template nucleic acid. During the S/G2 phase, an anti-HR inhibitor, as described herein, can prevent unproductive resolution of the HDR intermediate.

Accordingly, in some embodiments, the methods herein involve down-regulating the anti-HR pathway in order to promote HDR. For instance, the methods may involve modulating, e.g., inhibiting, a component (e.g., exactly one component, or one or more components, e.g., two or three components) of the anti-HR pathway, e.g., a component of Table VI.1(D) or Table VI.4. More specifically, the methods may involve modulating, e.g., inhibiting, exactly one of, or one or more of an anti-HR helicase, FBH1, RECQ5, BLM, a RecQ helicase, PARI, Rap80, FANCJ, FANCM, RECQ1, or RAD54. In embodiments, one or more of HDR, alt-HR, NHEJ, MMEJ, SSA, SSBR, MMR, NER, and BER are not substantially down-regulated, e.g., in some embodiments the only DNA damage repair pathway to be substantially down-regulated is the anti-HR pathway.

TABLE VI.4 Factors that promote anti-HR. Factor Sequence an anti-HR helicase — FBH1 >sp|Q8NFZ0|FBX18_HUMAN F-box only protein 18 OS = Homo sapiens GN = FBXO18 PE = 1 SV = 2 (SEQ ID NO: 220) MRRFKRKHLTAIDCQHLARSHLAVTQPFGQRWTNRDPNHGLYPKPRTKRGSRGQGSQRCI PEFFLAGKQPCTNDMAKSNSVGQDSCQDSEGDMIFPAESSCALPQEGSAGPGSPGSAPPS RKRSWSSEEESNQATGTSRWDGVSKKAPRHHLSVPCTRPREARQEAEDSTSRLSAESGET DQDAGDVGPDPIPDSYYGLLGTLPCQEALSHICSLPSEVLRHVFAFLPVEDLYWNLSLVC HLWREIISDPLFIPWKKLYHRYLMNEEQAVSKVDGILSNCGIEKESDLCVLNLIRYTATT KCSPSVDPERVLWSLRDHPLLPEAEACVRQHLPDLYAAAGGVNIWALVAAVVLLSSSVND IQRLLFCLRRPSSTVTMPDVTFTLYCIAVLLYAMREKGINISNRIHYNIFYCLYLQENSC TQATKVKEEPSVWPGKKTIQLTHEQQLILNHKMEPLQVVKIMAFAGTGKTSTLVKYAEKW SQSRFLYVTFNKSIAKQAERVFPSNVICKTFHSMAYGHIGRKYQSKKKLNLFKLTPFMVN SVLAEGKGGFIRAKLVCKTLENFFASADEELTIDHVPIWCKNSQGQRVMVEQSEKLNGVL EASRLWDNMRKLGECTEEAHQMTHDGYLKLWQLSKPSLASFDAIFVDEAQDCTPAIMNIV LSQPCGKIFVGDPHQQIYTFRGAVNALFTVPHTHVFYLTQSFRFGVEIAYVGATILDVCK RVRKKTLVGGNHQSGIRGDAKGQVALLSRTNANVFDEAVRVTEGEFPSRIHLIGGIKSFG LDRIIDIWILLQPEEERRKQNLVIKDKFIRRWVHKEGFSGFKRYVTAAEDKELEAKIAVV EKYNIRIPELVQRIEKCHIEDLDFAEYILGTVHKAKGLEFDTVHVLDDFVKVPCARHNLP QLPHFRVESFSEDEWNLLYVAVTRAKKRLIMTKSLENILTLAGEYFLQAELTSNVLKTGV VRCCVGQCNNAIPVDTVLTMKKLPITYSNRKENKGGYLCHSCAEQRIGPLAFLTASPEQV RAMERTVENIVLPRHEALLFLVF RECQ5 See Table VI.2 BLM See Table VI.2 FANCJ See Table VI.2 PARI >sp|Q9NWS1|PARI_HUMAN PCNA-interacting partner OS = Homo sapiens GN = PARPBP PE = 1 SV = 3 (SEQ ID NO: 221) MAVFNQKSVSDMIKEFRKNWRALCNSERTTLCGADSMLLALQLSMAENNKQHSGEFTVSL SDVLLTWKYLLHEKLNLPVENMDVTDHYEDVRKIYDDFLKNSNMLDLIDVYQKCRALTSN CENYNTVSPSQLLDFLSGKQYAVGDETDLSIPTSPTSKYNRDNEKVQLLARKIIFSYLNL LVNSKNDLAVAYILNIPDRGLGREAFTDLKHAAREKQMSIFLVATSFIRTIELGGKGYAP PPSDPLRTHVKGLSNFINFIDKLDEILGEIPNPSIAGGQILSVIKMQLIKGQNSRDPFCK AIEEVAQDLDLRIKNIINSQEGVVALSTTDISPARPKSHAINHGTAYCGRDTVKALLVLL DEEAANAPTKNKAELLYDEENTIHHHGTSILTLFRSPTQVNNSIKPLRERICVSMQEKKI KMKQTLIRSQFACTYKDDYMISKDNWNNVNLASKPLCVLYMENDLSEGVNPSVGRSTIGT SFGNVHLDRSKNEKVSRKSTSQTGNKSSKRKQVDLDGENILCDNRNEPPQHKNAKIPKKS NDSQNRLYGKLAKVAKSNKCTAKDKLISGQAKLTQFFRL >sp|Q9NWS1|PARI_HUMAN PCNA-interacting partner OS = Homo sapiens GN = PARPBP PE = 1 SV = 3 (SEQ ID NO: 222) MAVFNQKSVSDMIKEFRKNWRALCNSERTTLCGADSMLLALQLSMAENNKQHSGEFTVSL SDVLLTWKYLLHEKLNLPVENMDVTDHYEDVRKIYDDFLKNSNMLDLIDVYQKCRALTSN CENYNTVSPSQLLDFLSGKQYAVGDETDLSIPTSPTSKYNRDNEKVQLLARKIIFSYLNL LVNSKNDLAVAYILNIPDRGLGREAFTDLKHAAREKQMSIFLVATSFIRTIELGGKGYAP PPSDPLRTHVKGLSNFINFIDKLDEILGEIPNPSIAGGQILSVIKMQLIKGQNSRDPFCK AIEEVAQDLDLRIKNIINSQEGVVALSTTDISPARPKSHAINHGTAYCGRDTVKALLVLL DEEAANAPTKNKAELLYDEENTIHHHGTSILTLFRSPIQVNNSIKPLRERICVSMQEKKI KMKQTLIRSQFACTYKDDYMISKDNWNNVNLASKPLCVLYMENDLSEGVNPSVGRSTTGT SFGNVHLDRSKNEKVSRKSTSQTGNKSSKRKQVDLDGENILCDNRNEPPQHKNAKIPKKS NDSQNRLYGKLAKVAKSNKCTAKDKLISGQAKLTQFFRL >sp|Q9NWS1-2|PARI_HUMAN Isoform 2 of PCNA- interacting partner OS = Homo sapiens GN = PARPBP (SEQ ID NO: 223) MDVTDHYEDVRKIYDDFLKNSNMLDLIDVYQKCRALTSNCENYNTVSPSQLLDFLSGKQY AVGDETDLSIPTSPISKYNRDNEKVQLLARKIIFSYLNLLVNSKNDLAVAYILNIPDRGL GREAFTDLKHAAREKQMSIFLVATSFIRTIELGGKGYAPPPSDPLRTHVKGLSNFINFID KLDEILGEIPNPSIAGGQILSVIKMQLIKGQNSRDPFCKAIEEVAQDLDLRIKNIINSQE GVVALSTIDISPARPKSHAINHGTAYCGRDTVKALLVLLDEEAANAPTKNKAELLYDEEN TIHHHGTSILTLFRSPIQVNNSIKPLRERICVSMQEKKIKMKQTLIRSQFACTYKDDYMI SKDNWNNVNLASKPLCVLYMENDLSEGVNPSVGRSTIGTSFGNVHLDRSKNEKVSRKSTS QTGNKSSKRKQVDLDGENILCDNRNEPPQHKNAKIPKKSNDSQNRLYGKLAKVAKSNKCT AKDKLISGQAKLTQFFRL >sp|Q9NWS1-3|PARI_HUMAN Isoform 3 of PCNA- interacting partner OS = Homo sapiens GN = PARPBP (SEQ ID NO: 224) MAVFNQKSVSDMIKEFRKNWRALCNSERTTLCGADSMLLALQLSMAENNKQHSGEFTVSL SDVLLTWKYLLHEKLNLPVENMDVIDHYEDVRKIYDDFLKNSNMLDLIDVYQKCRALTSN CENYNTVSPSQLLDFLSGKQYAVGDETDLSIPTSPISKYNRDNEKALPVLKR >sp|Q9NWS1-4|PARI_HUMAN Isoform 4 of PCNA- interacting partner OS = Homo sapiens GN = PARPBP (SEQ ID NO: 225) MAVFNQKSVSDMIKEFRKNWRALCNSERTTLCGADSMLLALQLSMAENNKQHSGEFTVSL SDVLLTWKYLLHEKLNLPVENMDVIDHYEDVRKIYDDFLKNSNMLDLIDVYQKCRALTSN CENYNTVSPVSIF >sp|Q9NWS1-5|PARI_HUMAN Isoform 5 of PCNA- interacting partner OS = Homo sapiens GN = PARPBP (SEQ ID NO: 226) MQLIKGQNSRDPFCKAIEEVAQDLDLRIKNIINSQEGVVALSTIDISPARPKSHAINHGT AYCGRDTVKALLVLLDEEAANAPTKNKAELLYDEENTIHHHGTSILTLFRSPIQVNNSIK PLRERICVSMQEKKIKMKQTLIRSQFACTYKDDYMISKDNWNNVNLASKPLCVLYMENDL SEGVNPSVGRSTIGTSFGNVHLDRSKNEKVSRKSTSQTGNKSSKRKQVDLDGENILCDNR NEPPQHKNAKIPKKSNDSQNRLYGKLAKVAKSNKCIAKDKLISGQAKLTQFFRL >sp|Q9NWS1-6|PARI_HUMAN Isoform 6 of PCNA- interacting partner OS = Homo sapiens GN = PARPBP (SEQ ID NO: 227) MAVFNQKSVSDMIKEFRKNWRALCNSERTTLCGADSMLLALQLSMAENNKQHSGEFTVSL SDVLLTWKYLLHEKLNLPVENMDVIDHYEDVRKIYDDFLKNSNMLDLIDVYQKCRALTSN CENYNTVSPSQLLDFLSGKQYAVGDETDLSIPTSPISKYNRDNEKVQLLARKIIFSYLNL LVNSKNDLAVAYILNIPDRGLGREAFTDLKHAAREKQMSIFLVATSFIRTIELGGKGYAP PPSDPLRTHVKGLSNFINFIDKLDEILGEIPNPRGCKSICWKINNWNEFWKCSSGQK >sp|Q9NWS1-7|PARI_HUMAN Isoform 7 of PCNA- interacting partner OS = Homo sapiens GN = PARPBP (SEQ ID NO: 228) MAVFNQKSVSDMIKEFRKNWRALCNSERTTLCGADSMLLALQLSMAENNKQHSGEFTVSL SDVLLTWKYLLHEKLNLPVENMDVTDHYEDVRKIYDDFLKNSNMLDLIDVYQKCRALTSN CENYNTVSPSQLLDFLSGKQYAVGDETDLSIPTSPTSKYNRDNEKVQLLARKIIFSYLNL LVNSKNDLAVAYILNIPDRGLGREAFTDLKHAAREKQMSIFLVATSFIRTIELGGKGYAP PPSDPLRTHVKGLSNFINFIDKLDEILGEIPNPRSPTQVNNSIKPLRERICVSMQEKKIK RV RECQ1 See Table VI.2 WRN See Table VI.2 RTEL MPKIVLNGVTVDFPFQPYKCQQEYMTKVLECLQQKVNGILESPTGTGKTLCLLCTTLAWR EHLRDGISARKIAERAQGELFPDRALSSWGNAAAAAGDPIACYTDIPKIIYASRTHSQLT QVINELRNTSYRPKVCVLGSREQLCIHPEVKKQESNHLQIHLCRKKVASRSCHFYNNVEE KSLEQELASPILDIEDLVKSGSKHRVCPYYLSRNLKQQADIIFMPYNYLLDAKSRRAHNI DLKGTVVIFDEAHNVEKMCEESASFDLTPHDLASGLDVIDQVLEEQTKAAQQGEPHPEFS ADSPSPGLNMELEDIAKLKMILLRLEGAIDAVELPGDDSGVTKPGSYIFELFAEAQITFQ TKGCILDSLDQIIQHLAGRAGVFTNTAGLQKLADIIQIVFSVDPSEGSPGSPAGLGALQS YKVHIHPDAGHRRTAQRSDAWSTTAARKRGKVLSYWCFSPGHSMHELVRQGVRSLILTSG TLAPVSSFALEMQIPFPVCLENPHIIDKHQIWVGVVPRGPDGAQLSSAFDRRFSEECLSS LGKALGNIARVVPYGLLIFFPSYPVMEKSLEFWRARDLARKMEALKPLFVEPRSKGSFSE TISAYYARVAAPGSTGATFLAVCRGKASEGLDFSDTNGRGVIVTGLPYPPRMDPRVVLKM QFLDEMKGQGGAGGQFLSGQEWYRQQASRAVNQAIGRVIRHRQDYGAVFLCDHRFAFADA RAQLPSWVRPHVRVYDNFGHVIRDVAQFFRVAERTMPAPAPRATAPSVRGEDAVSEAKSP GPFFSTRKAKSLDLHVPSLKQRSSGSPAAGDPESSLCVEYEQEPVPARQRPRGLLAALEH SEQRAGSPGEEQAHSCSTLSLLSEKRPAEEPRGGRKKIRLVSHPEEPVAGAQTDRAKLFM VAVKQELSQANFATFTQALQDYKGSDDFAALAACLGPLFAEDPKKHNLLQGFYQFVRPHH KQQFEEVCIQLTGRGCGYRPEHSIPRRQRAQPVLDPTGRTAPDPKLTVSTAAAQQLDPQE HLNQGRPHLSPRPPPTGDPGSQPQWGSGVPRAGKQGQHAVSAYLADARRALGSAGCSQLL AALTAYKQDDDLDKVLAVLAALTTAKPEDFPLLHRFSMFVRPHHKQRFSQICIDLIGRPY PGMEPPGPQEERLAVPPVLTHRAPQPGPSRSEKTGKTQSKISSFLRQRPAGTVGAGGEDA GPSQSSGPPHGPAASEWGL (RTEL Isoform 1 CCDS 13531.1) (SEQ ID NO: 229) MPKIVLNGVTVDFPFQPYKCQQEYMTKVLECLQQKVNGILESPTGTGKTLCLLCTTLAWR EHLRDGISARKIAERAQGELFPDRALSSWGNAAAAAGDPIACYTDIPKIIYASRTHSQLT QVINELRNTSYRSRCRATLWVLETAPPRPTVLSPIRPKVCVLGSREQLCIHPEVKKQESN HLQIHLCRKKVASRSCHFYNNVEEKSLEQELASPILDIEDLVKSGSKHRVCPYYLSRNLK QQADIIFMPYNYLLDAKSRRAHNIDLKGTVVIFDEAHNVEKMCEESASFDLTPHDLASGL DVIDQVLEEQTKAAQQGEPHPEFSADSPSPGLNMELEDIAKLKMILLRLEGAIDAVELPG DDSGVTKPGSYIFELFAEAQITFQTKGCILDSLDQIIQHLAGRAGVFTNTAGLQKLADII QIVFSVDPSEGSPGSPAGLGALQSYKVHIHPDAGHRRTAQRSDAWSTTAARKRGKVLSYW CFSPGHSMHELVRQGVRSLILTSGTLAPVSSFALEMQIPFPVCLENPHIIDKHQIWVGVV PRGPDGAQLSSAFDRRFSEECLSSLGKALGNIARVVPYGLLIFFPSYPVMEKSLEFWRAR DLARKMEALKPLFVEPRSKGSFSETISAYYARVAAPGSTGATFLAVCRGKASEGLDFSDT NGRGVIVTGLPYPPRMDPRVVLKMQFLDEMKGQGGAGGQFLSGQEWYRQQASRAVNQAIG RVIRHRQDYGAVFLCDHRFAFADARAQLPSWVRPHVRVYDNFGHVIRDVAQFFRVAERTM PAPAPRATAPSVRGEDAVSEAKSPGPFFSTRKAKSLDLHVPSLKQRSSGSPAAGDPESSL CVEYEQEPVPARQRPRGLLAALEHSEQRAGSPGEEQAHSCSTLSLLSEKRPAEEPRGGRK KIRLVSHPEEPVAGAQTDRAKLFMVAVKQELSQANFATFTQALQDYKGSDDFAALAACLG PLFAEDPKKHNLLQGFYQFVRPHHKQQFEEVCIQLTGRGCGYRPEHSIPRRQRAQPVLDP TGRTAPDPKLTVSTAAAQQLDPQEHLNQGRPHLSPRPPPTGDPGSQPQWGSGVPRAGKQG QHAVSAYLADARRALGSAGCSQLLAALTAYKQDDDLDKVLAVLAALTTAKPEDFPLLHRF SMFVRPHHKQRFSQICIDLIGRPYPGMEPPGPQEERLAVPPVLTHRAPQPGPSRSEKTGK TQSKISSFLRQRPAGTVGAGGEDAGPSQSSGPPHGPAASEWGL (RTEL Isoform 2 CCDS 13530.3) (SEQ ID NO: 230) MPKIVLNGVTVDFPFQPYKCQQEYMTKVLECLQQKVNGILESPTGTGKTLCLLCTTLAWR EHLRDGISARKIAERAQGELFPDRALSSWGNAAAAAGDPIACYTDIPKIIYASRTHSQLT QVINELRNTSYRPKVCVLGSREQLCIHPEVKKQESNHLQIHLCRKKVASRSCHFYNNVEE KSLEQELASPILDIEDLVKSGSKHRVCPYYLSRNLKQQADIIFMPYNYLLDAKSRRAHNI DLKGTVVIFDEAHNVEKMCEESASFDLTPHDLASGLDVIDQVLEEQTKAAQQGEPHPEFS ADSPSPGLNMELEDIAKLKMILLRLEGAIDAVELPGDDSGVTKPGSYIFELFAEAQITFQ TKGCILDSLDQIIQHLAGRAGVFTNTAGLQKLADIIQIVFSVDPSEGSPGSPAGLGALQS YKVHIHPDAGHRRTAQRSDAWSTTAARKRGKVLSYWCFSPGHSMHELVRQGVRSLILTSG TLAPVSSFALEMQIPFPVCLENPHIIDKHQIWVGVVPRGPDGAQLSSAFDRRFSEECLSS LGKALGNIARVVPYGLLIFFPSYPVMEKSLEFWRARDLARKMEALKPLFVEPRSKGSFSE TISAYYARVAAPGSTGATFLAVCRGKASEGLDFSDTNGRGVIVTGLPYPPRMDPRVVLKM QFLDEMKGQGGAGGQFLSGQEWYRQQASRAVNQAIGRVIRHRQDYGAVFLCDHRFAFADA RAQLPSWVRPHVRVYDNFGHVIRDVAQFFRVAERTMPAPAPRATAPSVRGEDAVSEAKSP GPFFSTRKAKSLDLHVPSLKQRSSGSPAAGDPESSLCVEYEQEPVPARQRPRGLLAALEH SEQRAGSPGEEQAHSCSTLSLLSEKRPAEEPRGGRKKIRLVSHPEEPVAGAQTDRAKLFM VAVKQELSQANFATFTQALQDYKGSDDFAALAACLGPLFAEDPKKHNLLQGFYQFVRPHH KQQFEEVCIQLTGRGCGYRPEHSIPRRQRAQPVLDPTGRTAPDPKLTVSTAAAQQLDPQE HLNQGRPHLSPRPPPTGDPGSQPQWGSGVPRAGKQGQHAVSAYLADARRALGSAGCSQLL AALTAYKQDDDLDKVLAVLAALTTAKPEDFPLLHRFSMFVRPHHKQRFSQTCTDLTGRPY PGMEPPGPQEERLAVPPVLTHRAPQPGPSRSEKTGKTQSKISSFLRQRPAGTVGAGGEDA GPSQSSGPPHGPAASEWGEPHGRDIAGQQATGAPGGPLSAGCVCQGCGAEDVVPFQCPAC DFQRCQACWQRHLQASRMCPACHTASRKQSVMQVFWPEPQ (RTEL Isoform 3 CCDS 63331.1) (SEQ ID NO: 231) MPYNYLLDAKSRRAHNIDLKGTVVIFDEAHNVEKMCEESASFDLTPHDLASGLDVIDQVL EEQTKAAQQGEPHPEFSADSPSPGLNMELEDIAKLKMILLRLEGAIDAVELPGDDSGVTK PGSYIFELFAEAQITFQTKGCILDSLDQIIQHLAGRAGVFTNTAGLQKLADIIQIVFSVD PSEGSPGSPAGLGALQSYKVHIHPDAGHRRTAQRSDAWSTTAARKRGKVLSYWCFSPGHS MHELVRQGVRSLILTSGTLAPVSSFALEMQIPFPVCLENPHIIDKHQIWVGVVPRGPDGA QLSSAFDRRFSEECLSSLGKALGNIARVVPYGLLIFFPSYPVMEKSLEFWRARDLARKME ALKPLFVEPRSKGSFSETISAYYARVAAPGSTGATFLAVCRGKASEGLDFSDTNGRGVIV TGLPYPPRMDPRVVLKMQFLDEMKGQGGAGGQFLSGQEWYRQQASRAVNQAIGRVIRHRQ DYGAVFLCDHRFAFADARAQLPSWVRPHVRVYDNFGHVIRDVAQFFRVAERTMPAPAPRA TAPSVRGEDAVSEAKSPGPFFSTRKAKSLDLHVPSLKQRSSGSPAAGDPESSLCVEYEQE PVPARQRPRGLLAALEHSEQRAGSPGEEQAHSCSTLSLLSEKRPAEEPRGGRKKIRLVSH PEEPVAGAQTDRAKLFMVAVKQELSQANFATFTQALQDYKGSDDFAALAACLGPLFAEDP KKHNLLQGFYQFVRPHHKQQFEEVCIQLTGRGCGYRPEHSIPRRQRAQPVLDPTGRTAPD PKLTVSTAAAQQLDPQEHLNQGRPHLSPRPPPTGDPGSQPQWGSGVPRAGKQGQHAVSAY LADARRALGSAGCSQLLAALTAYKQDDDLDKVLAVLAALTTAKPEDFPLLHRFSMFVRPH HKQRFSQTCTDLTGRPYPGMEPPGPQEERLAVPPVLTHRAPQPGPSRSEKTGKTQSKISS FLRQRPAGTVGAGGEDAGPSQSSGPPHGPAASEWGL (RTEL Isoform 4 CCDS 74751.1) (SEQ ID NO: 232) Rap80 >sp|Q96RL1|UIMC1_HUMAN BRCA1-A complex subunit RAP80 OS = Homo sapiens GN = UIMC1 PE = 1 SV = 2 (SEQ ID NO: 233) MPRRKKKVKEVSESRNLEKKDVETTSSVSVKRKRRLEDAFIVISDSDGEEPKEENGLQKT KTKQSNRAKCLAKRKIAQMTEEEQFALALKMSEQEAREVNSQEEEEEELLRKAIAESLNS CRPSDASATRSRPLATGPSSQSHQEKTTDSGLTEGIWQLVPPSLFKGSHISQGNEAEERE EPWDHTEKTEEEPVSGSSGSWDQSSQPVFENVNVKSFDRCTGHSAEHTQCGKPQESTGRG SAFLKAVQGSGDTSRHCLPTLADAKGLQDTGGTVNYFWGIPFCPDGVDPNQYTKVILCQL EVYQKSLKMAQRQLLNKKGFGEPVLPRPPSLIQNECGQGEQASEKNECISEDMGDEDKEE RQESRASDWHSKTKDFQESSIKSLKEKLLLEEEPTTSHGQSSQGIVEETSEEGNSVPASQ SVAALTSKRSLVLMPESSAEEITVCPETQLSSSETFDLEREVSPGSRDILDGVRIIMADK EVGNKEDAEKEVAISTFSSSNQVSCPLCDQCFPPTKIERHAMYCNGLMEEDTVLIRRQKE AKTKSDSGTAAQTSLDIDKNEKCYLCKSLVPFREYQCHVDSCLQLAKADQGDGPEGSGRA CSTVEGKWQQRLKNPKEKGHSEGRLLSFLEQSEHKTSDADIKSSETGAFRVPSPGMEEAG CSREMQSSFTRRDLNESPVKSFVSISEATDCLVDFKKQVTVQPGSRTRTKAGRGRRRKF >sp|Q96RL1|UIMC1_HUMAN BRCA1-A complex subunit RAP80 OS = Homo sapiens GN = UIMC1 PE = 1 SV = 2 (SEQ ID NO: 234) MPRRKKKVKEVSESRNLEKKDVETTSSVSVKRKRRLEDAFIVISDSDGEEPKEENGLQKT KTKQSNRAKCLAKRKIAQMTEEEQFALALKMSEQEAREVNSQEEEEEELLRKAIAESLNS CRPSDASATRSRPLATGPSSQSHQEKTTDSGLTEGIWQLVPPSLFKGSHISQGNEAEERE EPWDHTEKTEEEPVSGSSGSWDQSSQPVFENVNVKSFDRCTGHSAEHTQCGKPQESTGRG SAFLKAVQGSGDTSRHCLPTLADAKGLQDTGGTVNYFWGIPFCPDGVDPNQYTKVILCQL EVYQKSLKMAQRQLLNKKGFGEPVLPRPPSLIQNECGQGEQASEKNECISEDMGDEDKEE RQESRASDWHSKTKDFQESSIKSLKEKLLLEEEPTTSHGQSSQGIVEETSEEGNSVPASQ SVAALTSKRSLVLMPESSAEEITVCPETQLSSSETFDLEREVSPGSRDILDGVRIIMADK EVGNKEDAEKEVAISTFSSSNQVSCPLCDQCFPPTKIERHAMYCNGLMEEDTVLIRRQKE AKTKSDSGTAAQTSLDIDKNEKCYLCKSLVPFREYQCHVDSCLQLAKADQGDGPEGSGRA CSTVEGKWQQRLKNPKEKGHSEGRLLSFLEQSEHKTSDADIKSSETGAFRVPSPGMEEAG CSREMQSSFTRRDLNESPVKSFVSISEATDCLVDFKKQVTVQPGSRTRTKAGRGRRRKF >sp|Q96RL1-2|UIMC1_HUMAN Isoform 2 of BRCA1- A complex subunit RAP80 OS = Homo sapiens GN = UIMC1 (SEQ ID NO: 235) MPRRKKKVKEVSESRNLEKKDVETTSSVSVKRKRRLEDAFIVISDSDGEEPKEENGLQKT KTKQSNRAKCLAKRKIAQMTEEEQFALALKMSEQEAREVNSQEEEEEELLRKAIAESLNS CRPSDASATRSRPLATGPSSQSHQEKTTDSGLTEGIWQLVPPSLFKGSHISQGNEAEERE EPWDHTEKTEEEPVSGSSGSWDQSSQPVFENVNVKSFDRCTGHSAEHTQCGKPQSSQGIV EETSEEGNSVPASQSVAALTSKRSLVLMPESSAEEITVCPETQLSSSETFDLEREVSPGS RDILDGVRIIMADKEVGNKEDAEKEVAISTFSSSNQVSCPLCDQCFPPTKIERHAMYCNG LMEEDTVLTRRQKEAKTKSDSGTAAQTSLDIDKNEKCYLCKSLVPFREYQCHVDSCLQLA KADQGDGPEGSGRACSTVEGKWQQRLKNPKEKGHSEGRLLSFLEQSEHKTSDADIKSSET GAFRVPSPGMEEAGCSREMQSSFTRRDLNESPVKSFVSISEATDCLVDFKKQVTVQPGSR TRTKAGRGRRRKF >sp|Q96RL1-3|UIMC1_HUMAN Isoform 3 of BRCA1- A complex subunit RAP80 OS = Homo sapiens GN = UIMC1 (SEQ ID NO: 236) MTEEEQFALALKMSEQEAREVNSQEEEEEELLRKAIAESLNSCRPSDASATRSRPLATGP SSQSHQEKTTDSGLTEGIWQLVPPSLFKGSHISQGNEAEEREEPWDHTEKTEEEPVSGSS GSWDQSSQPVFENVNVKSFDRCTGHSAEHTQCGKPQESTGRGSAFLKAVQGSGDTSRHCL PTLADAKGLQDTGGIVNYFWGIPFCPDGVDPNQYTKVILCQLEVYQKSLKMAQRQLLNKK GFGEPVLPRPPSLIQNECGQGEQASEKNECISEDMGDEDKEERQESRASDWHSKTKDFQE SSIKSLKEKLLLEEEPTTSHGQSSQGIVEETSEEGNSVPASQSVAALTSKRSLVLMPESS AEEITVCPETQLSSSETFDLEREVSPGSRDILDGVRIIMADKEVGNKEDAEKEVAISTFS SSNQVSCPLCDQCFPPTKIERHAMYCNGLMEEDTVLIRRQKEAKIKSDSGTAAQTSLDID KNEKCYLCKSLVPFREYQCHVDSCLQLAKADQGDGPEGSGRACSTVEGKWQQRLKNPKEK GHSEGRLLSFLEQSEHKTSDADIKSSETGAFRVPSPGMEEAGCSREMQSSFTRRDLNESP VKSFVSISEATDCLVDFKKQVTVQPGSRTRTKAGRGRRRKF >sp|Q96RL1-4|UIMC1_HUMAN Isoform 4 of BRCA1- A complex subunit RAP80 OS = Homo sapiens GN = UIMC1 (SEQ ID NO: 237) MLPLPDLDLWPLDRLPSPIKRKPQTLGSLKSSQGIVEETSEEGNSVPASQSVAALTSKRS LVLMPESSAEEITVCPETQLSSSETFDLEREVSPGSRDILDGVRIIMADKEVGNKEDAEK EVAISTFSSSNQVSCPLCDQCFPPTKIERHAMYCNGLMEEDTVLTRRQKEAKTKSDSGTA AQTSLDIDKNEKCYLCKSLVPFREYQCHVDSCLQLAKADQGDGPEGSGRACSTVEGKWQQ RLKNPKEKGHSEGRLLSFLEQSEHKTSDADIKSSETGAFRVPSPGMEEAGCSREMQSSFT RRDLNESPVKSFVSISEATDCLVDFKKQVTVQPGSRTRTKAGRGRRRKF >sp|Q96RL1-5|UIMC1_HUMAN Isoform 5 of BRCA1- A complex subunit RAP80 OS = Homo sapiens GN = UIMC1 (SEQ ID NO: 238) MPRRKKKVKEVSESRNLEKKDVETTSSVSVKRKRRLEDAFIVISDSDGEEPKEENGLQKT KTKQSNRAKCLAKRKIAQMTEEEQFALALKMSEQEAREVNSQEEEEEELLRKAIAESLNV NMPCCKSLWRLISYIFDFCGVVVALGTSCSHL miR-155 See Table VI.5 miR-545 See Table VI.5 miR-107 See Table VI.5 miR-1255 See Table VI.5 miR-148 See Table VI.5 miR-193 See Table VI.5

More specifically, in some embodiments, the down-regulator of an anti-HR pathway is an inhibitor of (e.g., an siRNA against) anti-HR helicase, FBH1, RECQ5, BLM, a RecQ helicase, PARM, Rap80, FANCJ, FANCM, RECQ, or RAD54, or an anti-miR that targets miR-15A, miR-545, miR-107, miR-1255, miR-148, or miR-193, or an agent of Table VI.5, or any combination thereof. In some embodiments, an anti-HR pathway is down regulated using an HDR-enhancing gRNA that targets a component of an anti-HR pathway, e.g., anti-HR helicase, FBH1, RECQ5, BLM, a RecQ helicase, PARI, Rap80, FANCJ, FANCM, RECQP, or RAD54. In some embodiments, the HDR-enhancing gRNA is used in combination with an eiCas9 molecule. In some embodiments, the HDR-enhancing gRNA is used in combination with an eaCas9 molecule. In some embodiments, the HDR-enhancing gRNA is administered to the cell as a HDR-enhancing gRNA:Cas9 molecule complex.

TABLE VI.5 Down-regulators of the anti-HR pathway. 1. Fbh1 siRNA FBXO18 Silencer, sold by Life Technologies FBXO18 F-box protein, helicase, 18 siRNA, sold by Dharmacon Commercially available from Dharmacon or Ambion antibodies Mouse Anti-Fbh1 antibody, monoclonal (2353C1a), sold by Lifespan Biosciences Anti-FBXO18/FBH1 antibody (aa748-777) LS-C166079, sold by Lifespan Biosciences 2. RTEL siRNA Commercially available from Dharmacon or Ambion 3. PARPI siRNA PMCH Silencer, sold by Life Technologies; PARPBP PARP1 binding protein siRNA, sold by Dharmacon; Commercially available from Dharmacon or Ambion antibodies PARP1 Binding Protein (PARPBP) (C-Term), (AA 522-550) antibody, sold by Aviva Systems Biology; PARP1 Binding Protein (PARPBP) (C-Term), (AA 522-550) antibody, sold by Atlas Antibodies 4. Rap80 siRNA UIMC1 Silencer, sold by Life Technologies; RAP80 siRNA (h), sold by Santa Cruz Biotechnology Entrez Entrez Gene Gene GenBank Reagent SEQ Symbol ID Acc. No. Source ID ID Sequence (sense) Rap 80 51720 NM_016290 D-006995-01 239 GAAAAUGGGUUGCAGAAAA Rap 80 51720 NM_016290 D-006995-03 240 AGAGGCAGCUCCUUAAUAA Rap 80 51720 NM_016290 D-006995-04 241 GCACAAGACUUCAGAUGCA Rap 80 51720 NM_016290 D-006995-05 242 GGACACAUCUAGGCACUGU antibodies RAP80 Antibody, sold by Bethyl Laboratories; Anti-RAP80 antibody (EPR5315) (ab124763), sold by Abcam; RAP80 (C-13) antibody, sold by Santa Cruz Biotechnology; RAP80 (E-17) antibody, sold by Santa Cruz Biotechnology; RAP80 (H-260) antibody, sold by Santa Cruz Biotechnology 5. Anti-miR for: mature miRNA Anti-miR sequence miR-155-5p UUAAUGCUAAUCGUGAUAGGGGU(243) ACCCCUAUCACGAUUAGCAUUAA(244) miR-155-3p CUCCUACAUAUUAGCAUUAACA(245) UGUUAAUGCUAAUAUGUAGGAG(246) miR-545-5p UCAGUAAAUGUUUAUUAGAUGA(247) UCAUCUAAUAAACAUUUACUGA(248) miR-545-3p UCAGCAAACAUUUAUUGUGUGC(249) GCACACAAUAAAUGUUUGCUGA(250) miR-107 AGCAGCAUUGUACAGGGCUAUCA(251) UGAUAGCCCUGUACAAUGCUGCU(252) miR-1255-A AGGAUGAGCAAAGAAAGUAGAUU(253) AAUCUACUUUCUUUGCUCAUCCU(254) miR-1255-B1 CGGAUGAGCAAAGAAAGUGGUU(255) AACCACUUUCUUUGCUCAUCCG(256) miR-1255-B2 CGGAUGAGCAAAGAAAGUGGUU(257) AACCACUUUCUUUGCUCAUCCG(258) miR-148-5p AAAGUUCUGAGACACUCCGACU(259) AGUCGGAGUGUCUCAGAACUUU(260) miR-148-3p UCAGUGCACUACAGAACUUUGU(261) ACAAAGUUCUGUAGUGCACUGA(262) miR-193-5p UGGGUCUUUGCGGGCGAGAUGA(263) UCAUCUCGCCCGCAAAGACCCA(264) miR-193-3p AACUGGCCUACAAAGUCCCAGU(265) ACUGGGACUUUGUAGGCCAGUU(266)

In other embodiments, the down-regulator of an HR pathway is an agent that promotes HDR by inhibiting resection. In one embodiment, agents that promotes HDR by inhibiting resection are listed in Table VI.6.

TABLE VI.6 Anti-HR Agents that Promote HDR by Inhibiting Resection 1) CtIP Entrez Entrez Gene Gene GenBank Reagent Source SEQ Symbol ID Acc. No. ID ID Sequence (sense) siRNA CtIP 5932 NM_002894 D-011376-01 267 GAGCAGACCUUUCUCAGUA CtIP 5932 NM_002894 D-011376-02 268 GAAGUGAACAAGAUCAUUA CtIP 5932 NM_002894 D-011376-03 269 CAACCAAGAUGUAUCCUUU CtIP 5932 NM_002894 D-011376-04 270 GAAUAGGACUGAGUACGGU CtIP 5932 NM_203292 J-011376-05 271 GGAGCUACCUCUAGUAUCA CtIP 5932 NM_203292 J-011376-06 272 GAGGUUAUAUUAAGGAAGA CtIP 5932 NM_203292 J-011376-07 273 GAACAGAAUAGGACUGAGU CtIP 5932 NM_203292 J-011376-08 274 GCACGUUGCCCAAAGAUUC CtIP 5932 NM_002894 s11849 275 UUACUUGUAAACCUUGUACtt CtIP 5932 NM_002894 s11850 276 AAAUCGAUCAGACAGAUCCag CtIP 5932 NM_002894 s11851 277 UCUUGUUCACUUCAGACCCaa 2) Exo1 siRNA Commercially available from Dharmacon or Ambion 3) DNA2 siRNA Commercially available from Dharmacon or Ambion 4) MRN complex Compounds Phase Mirin Phase I/II Telomelysin Resveratrol

VI.3 Canonical Non-Homologous End-Joining (Canonical NHEJ))

Canonical non-homologous end-joining is a second repair pathway that operates on double-stranded breaks. In contrast to HDR, canonical NHEJ typically acts when a DSB has blunt, unresected ends that are ligation-competent. The canonical NHEJ pathway can involve end processing i.e., deletions and/or fresh synthesis i.e., insertions. It can yield three types of different outcomes: correct repair (error free repair) or approximately 1 to 4 nucleotide insertions or deletions.

Canonical NHEJ depends on KU70-80 and Xrcc4/Ligase IV. Briefly, the core components of this pathway are KU70-80, DNApk, Artemis nuclease, DNA Polymerase, and ligase IV-XRCC4-XLF (these last 3 work as a complex to ligate ends). The pathway components are discussed below in more detail.

After a DSB forms, the toroidal heterodimer Ku70/Ku80 loads and then activates DNA-PKcs. DNA-PKcs stabilizes the DNA ends, preventing resection, and thus promotes canonical NHEJ over HDR and other DSB repair pathways. 53BP1 may also increase the stability of DSBs, thereby promoting canonical NHEJ. RIF1 and PTIP assist 53BP1 in this function. 53BP1 may also increase DSB mobility, allowing the DNA ends to find each other as is required for ligation. 53BP1, Rif1, and PTIP are not members of the core canonical NHEJ pathway. Rather, they act upstream of it, and regulate whether the core canonical NHEJ machinery engages at all. 53BP1, Rif1, and PTIP are anti-resection proteins.

To catalyze repair, XRCC4/LIG4 is recruited. If both ends are blunt and ligatable, a trimer of XRCC4, and LIG4 and XLF (a stimulatory factor) ligate the ends together. If ligation is not possible, an end-processing enzyme such as ARTEMIS or an APLF nuclease or the PNK kinase/phosphatase can transform the break into a substrate for ligation. Artemis, also known as DCLRElC (DNA cross-link repair 1C), has endonuclease activity on 5′ and 3′ overhangs, and a 5′ to 3′ exonuclease activity.

In some embodiments, HDR can be promoted by down-regulating a canonical NHEJ pathway. In some embodiments, a Cas9 molecule and gRNA can induce a DSB in a desired location during G1 or another phase of the cycle. This DSB can be formed using, e.g., one Cas9 molecule with the ability to produce DSBs, or two nickases. A canonical NHEJ down-regulator prevents repair of a DSB in G1. While in some cases that break might be repaired by, e.g., MMEJ, in other cases it will persist until S/G2, when HDR becomes active. A template nucleic acid can be added to the cell, so that the HDR machinery repairs the DSB using the template nucleic acid.

Accordingly, in some embodiments, the methods described herein involve down-regulating a canonical NHEJ pathway in order to promote HDR. For instance, the methods may involve modulating, e.g., inhibiting, a component (e.g., exactly one component, or one or more components, e.g., two or three components) of the canonical NHEJ pathway, e.g., a component of Table VI.7, VI.1(A), or VI.1(B). More specifically, in some embodiments, the methods described herein may involve modulating, e.g., inhibiting, exactly one of, or one or more of a Ku protein, Ku70, Ku80, DNA Pk, DNA-PKcs, 53BP1, XRCC4, LIG4, XLF, ARTEMIS, an APLF, or PNK. In some embodiments, the methods described herein may involve inhibiting an anti-resection protein, e.g., one or more of 53BP1, Rif1, and PTIP, in order to promote repair by HDR rather than canonical NHEJ. Up-regulation of HDR, in some embodiments, is achieved using siRNA directed against one or more of a Ku protein, Ku70, Ku80, DNA Pk, DNA-PKcs, 53BP1, XRCC4, LIG4, XLF, ARTEMIS, an APLF, PNK, Rif1, or PTIP. In some embodiments, a canonical NHEJ pathway is down regulated using an HDR-enhancing gRNA that targets a component of a canonical NHEJ pathway, e.g., one or more of a Ku protein, Ku70, Ku80, DNA Pk, DNA-PKcs, 53BP1, XRCC4, LIG4, XLF, ARTEMIS, an APLF, PNK, Rif1, or PTIP. In some embodiments, the HDR-enhancing gRNA is used in combination with an eiCas9 molecule. In some embodiments, the HDR-enhancing gRNA is used in combination with an eaCas9 molecule. In some embodiments, the HDR-enhancing gRNA is administered to the cell as a HDR-enhancing gRNA:Cas9 molecule complex. In another embodiment, the up-regulator of HDR is a dominant negative CtIP. A dominant negative CtIP promotes resection in G1 phase.

In some embodiments, one or more of HDR, alt-HR, anti-HR, MMEJ, SSA, SSBR, MMR, NER, and BER are not substantially down-regulated, e.g., the only DNA damage repair pathway to be substantially down-regulated is the canonical NHEJ pathway.

TABLE VI.7 Factors that promote canonical NHEJ Factor Sequence a Ku protein — Ku70 (also called >sp|P12956|XRCC6_HUMAN X-ray repair cross-complementing protein 6 XRCC6) OS = Homo sapiens GN = XRCC6 PE = 1 SV = 2 (SEQ ID NO: 278) MSGWESYYKTEGDEEAEEEQEENLEASGDYKYSGRDSLIFLVDASKAMFESQSEDELTPF DMSIQCIQSVYISKIISSDRDLLAVVFYGTEKDKNSVNFKNIYVLQELDNPGAKRILELD QFKGQQGQKRFQDMMGHGSDYSLSEVLWVCANLFSDVQFKMSHKRIMLFTNEDNPHGNDS AKASRARTKAGDLRDTGIFLDLMHLKKPGGFDISLFYRDIISIAEDEDLRVHFEESSKLE DLLRKVRAKETRKRALSRLKLKLNKDIVISVGIYNLVQKALKPPPIKLYRETNEPVKTKT RTFNTSTGGLLLPSDTKRSQIYGSRQIILEKEETEELKRFDDPGLMLMGFKPLVLLKKHH YLRPSLFVYPEESLVIGSSTLFSALLIKCLEKEVAALCRYTPRRNIPPYFVALVPQEEEL DDQKIQVTPPGFQLVFLPFADDKRKMPFTEKIMATPEQVGKMKAIVEKLRFTYRSDSFEN PVLQQHFRNLEALALDLMEPEQAVDLTLPKVEAMNKRLGSLVDEFKELVYPPDYNPEGKV TKRKHDNEGSGSKRPKVEYSEEELKTHISKGTLGKFTVPMLKEACRAYGLKSGLKKQELL EALTKHFQD >sp|P12956-2|XRCC6_HUMAN Isoform 2 of X-ray repair cross- complementing protein 6 OS = Homo sapiens GN = XRCC6 (SEQ ID NO: 279) MSGWESYYKTEGDEEAEEEQEENLEASGDYKYSGRDSLIFLVDASKAMFESQSEDELTPF DMSIQELDNPGAKRILELDQFKGQQGQKRFQDMMGHGSDYSLSEVLWVCANLFSDVQFKM SHKRIMLFTNEDNPHGNDSAKASRARTKAGDLRDTGIFLDLMHLKKPGGFDISLFYRDII SIAEDEDLRVHFEESSKLEDLLRKVRAKETRKRALSRLKLKLNKDIVISVGIYNLVQKAL KPPPIKLYRETNEPVKIKTRIFNISIGGLLLPSDTKRSQIYGSRQIILEKEETEELKRFD DPGLMLMGFKPLVLLKKHHYLRPSLFVYPEESLVIGSSTLFSALLIKCLEKEVAALCRYT PRRNIPPYFVALVPQEEELDDQKIQVIPPGFQLVFLPFADDKRKMPFTEKIMATPEQVGK MKAIVEKLRFTYRSDSFENPVLQQHFRNLEALALDLMEPEQAVDLTLPKVEAMNKRLGSL VDEFKELVYPPDYNPEGKVIKRKHDNEGSGSKRPKVEYSEEELKTHISKGILGKFTVPML KEACRAYGLKSGLKKQELLEALTKHFQD Ku80 >sp|P13010|XRCC5_HUMAN X-ray repair cross-complementing protein 5 OS = Homo sapiens GN = XRCC5 PE = 1 SV = 3 (SEQ ID NO: 280) MVRSGNKAAVVLCMDVGFTMSNSIPGIESPFEQAKKVITMFVQRQVFAENKDEIALVLFG TDGIDNPLSGGDQYQNITVHRHLMLPDFDLLEDIESKIQPGSQQADFLDALIVSMDVIQH ETIGKKFEKRHIEIFIDLSSRFSKSQLDIIIHSLKKCDISLQFFLPFSLGKEDGSGDRGD GPFRLGGHGPSFPLKGITEQQKEGLEIVKMVMISLEGEDGLDEIYSFSESLRKLCVFKKI ERHSIHWPCRLTIGSNLSIRIAAYKSILQERVKKTWTVVDAKTLKKEDIQKETVYCLNDD DETEVLKEDIIQGFRYGSDIVPFSKVDEEQMKYKSEGKCFSVLGFCKSSQVQRRFFMGNQ VLKVFAARDDEAAAVALSSLIHALDDLDMVAIVRYAYDKRANPQVGVAFPHIKHNYECLV YVQLPFMEDLRQYMFSSLKNSKKYAPTEAQLNAVDALIDSMSLAKKDEKTDTLEDLFPTT KIPNPRFQRLFQCLLHRALHPREPLPPIQQHIWNMLNPPAEVTTKSQIPLSKIKTLFPLI EAKKKDQVTAQEIFQDNHEDGPTAKKLKTEQGGAHFSVSSLAEGSVTSVGSVNPAENFRV LVKQKKASFEEASNQLINHIEQFLDTNETPYFMKSIDCIRAFREEAIKFSEEQRFNNFLK ALQEKVEIKQLNHFWEIVVQDGITLITKEEASGSSVTAEEAKKFLAPKDKPSGDTAAVFE EGGDVDDLLDMI DNA Pk — DNA-PKcs >sp|P78527|PRKDC_HUMAN DNA-dependent protein kinase catalytic subunit OS = Homo sapiens GN = PRKDC PE = 1 SV = 3 (SEQ ID NO: 281) MAGSGAGVRCSLLRLQETLSAADRCGAALAGHQLIRGLGQECVLSSSPAVLALQTSLVFS RDFGLLVFVRKSLNSIEFRECREEILKFLCIFLEKMGQKIAPYSVEIKNTCTSVYTKDRA AKCKIPALDLLIKLLQTFRSSRLMDEFKIGELFSKFYGELALKKKIPDTVLEKVYELLGL LGEVHPSEMINNAENLFRAFLGELKTQMTSAVREPKLPVLAGCLKGLSSLLCNFTKSMEE DPQTSREIFNFVLKAIRPQIDLKRYAVPSAGLRLFALHASQFSTCLLDNYVSLFEVLLKW CAHTNVELKKAALSALESFLKQVSNMVAKNAEMHKNKLQYFMEQFYGIIRNVDSNNKELS IAIRGYGLFAGPCKVINAKDVDFMYVELIQRCKQMFLTQTDTGDDRVYQMPSFLQSVASV LLYLDTVPEVYTPVLEHLVVMQIDSFPQYSPKMQLVCCRAIVKVFLALAAKGPVLRNCIS TVVHQGLIRICSKPVVLPKGPESESEDHRASGEVRTGKWKVPTYKDYVDLFRHLLSSDQM MDSILADEAFFSVNSSSESLNHLLYDEFVKSVLKIVEKLDLTLEIQTVGEQENGDEAPGV WMIPTSDPAANLHPAKPKDFSAFINLVEFCREILPEKQAEFFEPWVYSFSYELILQSTRL PLISGFYKLLSITVRNAKKIKYFEGVSPKSLKHSPEDPEKYSCFALFVKFGKEVAVKMKQ YKDELLASCLTFLLSLPHNIIELDVRAYVPALQMAFKLGLSYTPLAEVGLNALEEWSIYI DRHVMQPYYKDILPCLDGYLKTSALSDETKNNWEVSALSRAAQKGFNKVVLKHLKKTKNL SSNEAISLEEIRIRVVQMLGSLGGQINKNLLTVTSSDEMMKSYVAWDREKRLSFAVPFRE MKPVIFLDVFLPRVTELALTASDRQTKVAACELLHSMVMFMLGKATQMPEGGQGAPPMYQ LYKRTFPVLLRLACDVDQVTRQLYEPLVMQLIHWFTNNKKFESQDTVALLEAILDGIVDP VDSTLRDFCGRCIREFLKWSIKQITPQQQEKSPVNTKSLFKRLYSLALHPNAFKRLGASL AFNNIYREFREEESLVEQFVFEALVIYMESLALAHADEKSLGTIQQCCDAIDHLCRIIEK KHVSLNKAKKRRLPRGFPPSASLCLLDLVKWLLAHCGRPQTECRHKSIELFYKFVPLLPG NRSPNLWLKDVLKEEGVSFLINTFEGGGCGQPSGILAQPTLLYLRGPFSLQATLCWLDLL LAALECYNTFIGERTVGALQVLGTEAQSSLLKAVAFFLESIAMHDIIAAEKCFGTGAAGN RTSPQEGERYNYSKCTVVVRIMEFTTTLLNTSPEGWKLLKKDLCNTHLMRVLVQTLCEPA SIGFNIGDVQVMAHLPDVCVNLMKALKMSPYKDILETHLREKITAQSIEELCAVNLYGPD AQVDRSRLAAVVSACKQLHRAGLLHNILPSQSTDLHHSVGTELLSLVYKGIAPGDERQCL PSLDLSCKQLASGLLELAFAFGGLCERLVSLLLNPAVLSTASLGSSQGSVIHFSHGEYFY SLFSETINTELLKNLDLAVLELMQSSVDNTKMVSAVLNGMLDQSFRERANQKHQGLKLAT TILQHWKKCDSWWAKDSPLETKMAVLALLAKILQIDSSVSFNTSHGSFPEVFTTYISLLA DTKLDLHLKGQAVTLLPFFTSLTGGSLEELRRVLEQLIVAHFPMQSREFPPGTPRFNNYV DCMKKFLDALELSQSPMLLELMTEVLCREQQHVMEELFQSSFRRIARRGSCVTQVGLLES VYEMFRKDDPRLSFTRQSFVDRSLLTLLWHCSLDALREFFSTIVVDAIDVLKSRFTKLNE STFDTQITKKMGYYKILDVMYSRLPKDDVHAKESKINQVFHGSCITEGNELTKTLIKLCY DAFTENMAGENQLLERRRLYHCAAYNCAISVICCVFNELKFYQGFLFSEKPEKNLLIFEN LIDLKRRYNFPVEVEVPMERKKKYIEIRKEAREAANGDSDGPSYMSSLSYLADSTLSEEM SQFDFSTGVQSYSYSSQDPRPATGRFRRREQRDPTVHDDVLELEMDELNRHECMAPLTAL VKHMHRSLGPPQGEEDSVPRDLPSWMKFLHGKLGNPIVPLNIRLFLAKLVINTEEVFRPY AKHWLSPLLQLAASENNGGEGIHYMVVEIVATILSWTGLATPTGVPKDEVLANRLLNFLM KHVFHPKRAVFRHNLEIIKTLVECWKDCLSIPYRLIFEKFSGKDPNSKDNSVGIQLLGIV MANDLPPYDPQCGIQSSEYFQALVNNMSFVRYKEVYAAAAEVLGLILRYVMERKNILEES LCELVAKQLKQHQNTMEDKFIVCLNKVTKSFPPLADRFMNAVFFLLPKFHGVLKTLCLEV VLCRVEGMTELYFQLKSKDFVQVMRHRDDERQKVCLDIIYKMMPKLKPVELRELLNPVVE FVSHPSTTCREQMYNILMWIHDNYRDPESETDNDSQEIFKLAKDVLIQGLIDENPGLQLI IRNFWSHETRLPSNTLDRLLALNSLYSPKIEVHFLSLATNFLLEMTSMSPDYPNPMFEHP LSECEFQEYTIDSDWRFRSTVLTPMFVETQASQGTLQTRTQEGSLSARWPVAGQIRATQQ QHDFTLTQTADGRSSFDWLTGSSTDPLVDHTSPSSDSLLFAHKRSERLQRAPLKSVGPDF GKKRLGLPGDEVDNKVKGAAGRTDLLRLRRRFMRDQEKLSLMYARKGVAEQKREKEIKSE LKMKQDAQVVLYRSYRHGDLPDIQIKHSSLITPLQAVAQRDPIIAKQLFSSLFSGILKEM DKFKTLSEKNNITQKLLQDFNRFLNTTFSFFPPFVSCIQDISCQHAALLSLDPAAVSAGC LASLQQPVGIRLLEEALLRLLPAELPAKRVRGKARLPPDVLRWVELAKLYRSIGEYDVLR GIFTSEIGTKQITQSALLAEARSDYSEAAKQYDEALNKQDWVDGEPTEAEKDFWELASLD CYNHLAEWKSLEYCSTASIDSENPPDLNKIWSEPFYQETYLPYMIRSKLKLLLQGEADQS LLTFIDKAMHGELQKAILELHYSQELSLLYLLQDDVDRAKYYIQNGIQSFMQNYSSIDVL LHQSRLTKLQSVQALTEIQEFISFISKQGNLSSQVPLKRLLNTWTNRYPDAKMDPMNIWD DIITNRCFFLSKIEEKLTPLPEDNSMNVDQDGDPSDRMEVQEQEEDISSLIRSCKFSMKM KMIDSARKQNNFSLAMKLLKELHKESKTRDDWLVSWVQSYCRLSHCRSRSQGCSEQVLTV LKTVSLLDENNVSSYLSKNILAFRDQNILLGTTYRIIANALSSEPACLAEIEEDKARRIL ELSGSSSEDSEKVIAGLYQRAFQHLSEAVQAAEEEAQPPSWSCGPAAGVIDAYMTLADFC DQQLRKEEENASVIDSAELQAYPALVVEKMLKALKLNSNEARLKFPRLLQIIERYPEETL SLMTKEISSVPCWQFISWISHMVALLDKDQAVAVQHSVEEITDNYPQAIVYPFIISSESY SFKDTSTGHKNKEFVARIKSKLDQGGVIQDFINALDQLSNPELLFKDWSNDVRAELAKTP VNKKNIEKMYERMYAALGDPKAPGLGAFRRKFIQTFGKEFDKHFGKGGSKLLRMKLSDFN DITNMLLLKMNKDSKPPGNLKECSPWMSDFKVEFLRNELEIPGQYDGRGKPLPEYHVRIA GFDERVTVMASLRRPKRIIIRGHDEREHPFLVKGGEDLRQDQRVEQLFQVMNGILAQDSA CSQRALQLRTYSVVPMTSRLGLIEWLENTVTLKDLLLNTMSQEEKAAYLSDPRAPPCEYK DWLTKMSGKHDVGAYMLMYKGANRTETVTSFRKRESKVPADLLKRAFVRMSTSPEAFLAL RSHFASSHALICISHWILGIGDRHLNNFMVAMETGGVIGIDFGHAFGSATQFLPVPELMP FRLTRQFINLMLPMKETGLMYSIMVHALRAFRSDPGLLTNTMDVFVKEPSFDWKNFEQKM LKKGGSWIQEINVAEKNWYPRQKICYAKRKLAGANPAVITCDELLLGHEKAPAFRDYVAV ARGSKDHNIRAQEPESGLSEETQVKCLMDQATDPNILGRTWEGWEPWM >sp|P78527-2|PRKDC_HUMAN Isoform 2 of DNA-dependent protein kinase catalytic subunit OS = Homo sapiens GN = PRKDC (SEQ ID NO: 282) MAGSGAGVRCSLLRLQETLSAADRCGAALAGHQLIRGLGQECVLSSSPAVLALQTSLVFS RDFGLLVFVRKSLNSIEFRECREEILKFLCIFLEKMGQKIAPYSVEIKNTCTSVYTKDRA AKCKIPALDLLIKLLQTFRSSRLMDEFKIGELFSKFYGELALKKKIPDTVLEKVYELLGL LGEVHPSEMINNAENLFRAFLGELKTQMTSAVREPKLPVLAGCLKGLSSLLCNFTKSMEE DPQTSREIFNFVLKAIRPQIDLKRYAVPSAGLRLFALHASQFSTCLLDNYVSLFEVLLKW CAHTNVELKKAALSALESFLKQVSNMVAKNAEMHKNKLQYFMEQFYGIIRNVDSNNKELS IAIRGYGLFAGPCKVINAKDVDFMYVELIQRCKQMFLTQTDTGDDRVYQMPSFLQSVASV LLYLDTVPEVYTPVLEHLVVMQIDSFPQYSPKMQLVCCRAIVKVFLALAAKGPVLRNCIS TVVHQGLIRICSKPVVLPKGPESESEDHRASGEVRTGKWKVPTYKDYVDLFRHLLSSDQM MDSILADEAFFSVNSSSESLNHLLYDEFVKSVLKIVEKLDLTLEIQTVGEQENGDEAPGV WMIPTSDPAANLHPAKPKDFSAFINLVEFCREILPEKQAEFFEPWVYSFSYELILQSTRL PLISGFYKLLSITVRNAKKIKYFEGVSPKSLKHSPEDPEKYSCFALFVKFGKEVAVKMKQ YKDELLASCLTFLLSLPHNIIELDVRAYVPALQMAFKLGLSYTPLAEVGLNALEEWSIYI DRHVMQPYYKDILPCLDGYLKTSALSDETKNNWEVSALSRAAQKGFNKVVLKHLKKTKNL SSNEAISLEEIRIRVVQMLGSLGGQINKNLLTVTSSDEMMKSYVAWDREKRLSFAVPFRE MKPVIFLDVFLPRVTELALTASDRQTKVAACELLHSMVMFMLGKATQMPEGGQGAPPMYQ LYKRTFPVLLRLACDVDQVTRQLYEPLVMQLIHWFTNNKKFESQDTVALLEAILDGIVDP VDSTLRDFCGRCIREFLKWSIKQITPQQQEKSPVNTKSLFKRLYSLALHPNAFKRLGASL AFNNIYREFREEESLVEQFVFEALVIYMESLALAHADEKSLGTIQQCCDAIDHLCRIIEK KHVSLNKAKKRRLPRGFPPSASLCLLDLVKWLLAHCGRPQTECRHKSIELFYKFVPLLPG NRSPNLWLKDVLKEEGVSFLINTFEGGGCGQPSGILAQPTLLYLRGPFSLQATLCWLDLL LAALECYNTFIGERTVGALQVLGTEAQSSLLKAVAFFLESIAMHDIIAAEKCFGTGAAGN RTSPQEGERYNYSKCTVVVRIMEFTTTLLNTSPEGWKLLKKDLCNTHLMRVLVQTLCEPA SIGFNIGDVQVMAHLPDVCVNLMKALKMSPYKDILETHLREKITAQSIEELCAVNLYGPD AQVDRSRLAAVVSACKQLHRAGLLHNILPSQSTDLHHSVGTELLSLVYKGIAPGDERQCL PSLDLSCKQLASGLLELAFAFGGLCERLVSLLLNPAVLSTASLGSSQGSVIHFSHGEYFY SLFSETINTELLKNLDLAVLELMQSSVDNTKMVSAVLNGMLDQSFRERANQKHQGLKLAT TILQHWKKCDSWWAKDSPLETKMAVLALLAKILQIDSSVSFNTSHGSFPEVFTTYISLLA DTKLDLHLKGQAVTLLPFFTSLTGGSLEELRRVLEQLIVAHFPMQSREFPPGTPRFNNYV DCMKKFLDALELSQSPMLLELMTEVLCREQQHVMEELFQSSFRRIARRGSCVTQVGLLES VYEMFRKDDPRLSFTRQSFVDRSLLTLLWHCSLDALREFFSTIVVDAIDVLKSRFTKLNE STFDTQITKKMGYYKILDVMYSRLPKDDVHAKESKINQVFHGSCITEGNELTKTLIKLCY DAFTENMAGENQLLERRRLYHCAAYNCAISVICCVFNELKFYQGFLFSEKPEKNLLIFEN LIDLKRRYNFPVEVEVPMERKKKYIEIRKEAREAANGDSDGPSYMSSLSYLADSTLSEEM SQFDFSTGVQSYSYSSQDPRPATGRFRRREQRDPTVHDDVLELEMDELNRHECMAPLTAL VKHMHRSLGPPQGEEDSVPRDLPSWMKFLHGKLGNPIVPLNIRLFLAKLVINTEEVFRPY AKHWLSPLLQLAASENNGGEGIHYMVVEIVATILSWTGLATPTGVPKDEVLANRLLNFLM KHVFHPKRAVFRHNLEIIKTLVECWKDCLSIPYRLIFEKFSGKDPNSKDNSVGIQLLGIV MANDLPPYDPQCGIQSSEYFQALVNNMSFVRYKEVYAAAAEVLGLILRYVMERKNILEES LCELVAKQLKQHQNTMEDKFIVCLNKVTKSFPPLADRFMNAVFFLLPKFHGVLKTLCLEV VLCRVEGMTELYFQLKSKDFVQVMRHRDDERQKVCLDIIYKMMPKLKPVELRELLNPVVE FVSHPSTTCREQMYNILMWIHDNYRDPESETDNDSQEIFKLAKDVLIQGLIDENPGLQLI IRNFWSHETRLPSNTLDRLLALNSLYSPKIEVHFLSLATNFLLEMTSMSPDYPNPMFEHP LSECEFQEYTIDSDWRFRSTVLTPMFVETQASQGTLQTRTQEGSLSARWPVAGQIRATQQ QHDFTLTQTADGRSSFDWLTGSSTDPLVDHTSPSSDSLLFAHKRSERLQRAPLKSVGPDF GKKRLGLPGDEVDNKVKGAAGRTDLLRLRRRFMRDQEKLSLMYARKGVAEQKREKEIKSE LKMKQDAQVVLYRSYRHGDLPDIQIKHSSLITPLQAVAQRDPIIAKQLFSSLFSGILKEM DKFKTLSEKNNITQKLLQDFNRFLNTTFSFFPPFVSCIQDISCQHAALLSLDPAAVSAGC LASLQQPVGIRLLEEALLRLLPAELPAKRVRGKARLPPDVLRWVELAKLYRSIGEYDVLR GIFTSEIGTKQITQSALLAEARSDYSEAAKQYDEALNKQDWVDGEPTEAEKDFWELASLD CYNHLAEWKSLEYCSTASIDSENPPDLNKIWSEPFYQETYLPYMIRSKLKLLLQGEADQS LLTFIDKAMHGELQKAILELHYSQELSLLYLLQDDVDRAKYYIQNGIQSFMQNYSSIDVL LHQSRLTKLQSVQALTEIQEFISFISKQGNLSSQVPLKRLLNTWTNRYPDAKMDPMNIWD DIITNRCFFLSKIEEKLTPLPEDNSMNVDQDGDPSDRMEVQEQEEDISSLIRSCKFSMKM KMIDSARKQNNFSLAMKLLKELHKESKTRDDWLVSWVQSYCRLSHCRSRSQGCSEQVLTV LKTVSLLDENNVSSYLSKNILAFRDQNILLGTTYRIIANALSSEPACLAEIEEDKARRIL ELSGSSSEDSEKVIAGLYQRAFQHLSEAVQAAEEEAQPPSWSCGPAAGVIDAYMTLADFC DQQLRKEEENASVIDSAELQAYPALVVEKMLKALKLNSNEARLKFPRLLQIIERYPEETL SLMTKEISSVPCWQFISWISHMVALLDKDQAVAVQHSVEEITDNYPQAIVYPFIISSESY SFKDTSTGHKNKEFVARIKSKLDQGGVIQDFINALDQLSNPELLFKDWSNDVRAELAKTP VNKKNIEKMYERMYAALGDPKAPGLGAFRRKFIQTFGKEFDKHFGKGGSKLLRMKLSDFN DITNMLLLKMNKDSKPPGNLKECSPWMSDFKVEFLRNELEIPGQYDGRGKPLPEYHVRIA GFDERVTVMASLRRPKRIIIRGHDEREHPFLVKGGEDLRQDQRVEQLFQVMNGILAQDSA CSQRALQLRTYSVVPMTSSDPRAPPCEYKDWLTKMSGKHDVGAYMLMYKGANRTETVTSF RKRESKVPADLLKRAFVRMSTSPEAFLALRSHFASSHALICISHWILGIGDRHLNNFMVA METGGVIGIDFGHAFGSATQFLPVPELMPFRLTRQFINLMLPMKETGLMYSIMVHALRAF RSDPGLLTNTMDVFVKEPSFDWKNFEQKMLKKGGSWIQEINVAEKNWYPRQKICYAKRKL AGANPAVITCDELLLGHEKAPAFRDYVAVARGSKDHNIRAQEPESGLSEETQVKCLMDQA TDPNILGRTWEGWEPWM 53BP1 >sp|Q12888|TP53B_HUMAN Tumor suppressor p53-binding protein 1 OS = Homo sapiens GN = TP53BP1 PE = 1 SV = 2 (SEQ ID NO: 283) MDPTGSQLDSDFSQQDTPCLIIEDSQPESQVLEDDSGSHFSMLSRHLPNLQTHKENPVLD VVSNPEQTAGEERGDGNSGFNEHLKENKVADPVDSSNLDTCGSISQVIEQLPQPNRTSSV LGMSVESAPAVEEEKGEELEQKEKEKEEDTSGNTTHSLGAEDTASSQLGFGVLELSQSQD VEENTVPYEVDKEQLQSVTTNSGYTRLSDVDANTAIKHEEQSNEDIPIAEQSSKDIPVTA QPSKDVHVVKEQNPPPARSEDMPFSPKASVAAMEAKEQLSAQELMESGLQIQKSPEPEVL STQEDLFDQSNKTVSSDGCSTPSREEGGCSLASTPATTLHLLQLSGQRSLVQDSLSTNSS DLVAPSPDAFRSTPFIVPSSPTEQEGRQDKPMDTSVLSEEGGEPFQKKLQSGEPVELENP PLLPESTVSPQASTPISQSTPVFPPGSLPIPSQPQFSHDIFIPSPSLEEQSNDGKKDGDM HSSSLTVECSKTSEIEPKNSPEDLGLSLTGDSCKLMLSTSEYSQSPKMESLSSHRIDEDG ENTQIEDTEPMSPVLNSKFVPAENDSILMNPAQDGEVQLSQNDDKTKGDDTDTRDDISIL ATGCKGREETVAEDVCIDLTCDSGSQAVPSPATRSEALSSVLDQEEAMEIKEHHPEEGSS GSEVEEIPETPCESQGEELKEENMESVPLHLSLTETQSQGLCLQKEMPKKECSEAMEVET SVISIDSPQKLAILDQELEHKEQEAWEEATSEDSSVVIVDVKEPSPRVDVSCEPLEGVEK CSDSQSWEDIAPEIEPCAENRLDTKEEKSVEYEGDLKSGTAETEPVEQDSSQPSLPLVRA DDPLRLDQELQQPQTQEKTSNSLTEDSKMANAKQLSSDAEAQKLGKPSAHASQSFCESSS ETPFHFTLPKEGDIIPPLTGATPPLIGHLKLEPKRHSTPIGISNYPESTIATSDVMSESM VETHDPILGSGKGDSGAAPDVDDKLCLRMKLVSPETEASEESLQFNLEKPATGERKNGST AVAESVASPQKTMSVLSCICEARQENEARSEDPPTTPIRGNLLHFPSSQGEEEKEKLEGD HTIRQSQQPMKPISPVKDPVSPASQKMVIQGPSSPQGEAMVTDVLEDQKEGRSTNKENPS KALIERPSQNNIGIQTMECSLRVPETVSAATQTIKNVCEQGTSTVDQNFGKQDATVQTER GSGEKPVSAPGDDTESLHSQGEEEFDMPQPPHGHVLHRHMRTIREVRTLVTRVITDVYYV DGTEVERKVTEETEEPIVECQECETEVSPSQTGGSSGDLGDISSFSSKASSLHRTSSGTS LSAMHSSGSSGKGAGPLRGKTSGTEPADFALPSSRGGPGKLSPRKGVSQTGTPVCEEDGD AGLGIRQGGKAPVTPRGRGRRGRPPSRTTGTRETAVPGPLGIEDISPNLSPDDKSFSRVV PRVPDSTRRTDVGAGALRRSDSPEIPFQAAAGPSDGLDASSPGNSFVGLRVVAKWSSNGY FYSGKITRDVGAGKYKLLFDDGYECDVLGKDILLCDPIPLDTEVTALSEDEYFSAGVVKG HRKESGELYYSIEKEGQRKWYKRMAVILSLEQGNRLREQYGLGPYEAVTPLTKAADISLD NLVEGKRKRRSNVSSPATPTASSSSSTTPTRKITESPRASMGVLSGKRKLITSEEERSPA KRGRKSATVKPGAVGAGEFVSPCESGDNTGEPSALEEQRGPLPLNKTLFLGYAFLLTMAT TSDKLASRSKLPDGPTGSSEEEEEFLEIPPFNKQYTESQLRAGAGYILEDFNEAQCNTAY QCLLIADQHCRTRKYFLCLASGIPCVSHVWVHDSCHANQLQNYRNYLLPAGYSLEEQRIL DWQPRENPFQNLKVLLVSDQQQNFLELWSEILMTGGAASVKQHHSSAHNKDIALGVFDVV VTDPSCPASVLKCAEALQLPVVSQEWVIQCLIVGERIGFKQHPKYKHDYVSH >sp|Q12888-2|TP53B_HUMAN Isoform 2 of Tumor suppressor p53- binding protein 1 OS = Homo sapiens GN = TP53BP1 (SEQ ID NO: 284) MPGEQMDPTGSQLDSDFSQQDTPCLIIEDSQPESQVLEDDSGSHFSMLSRHLPNLQTHKE NPVLDVVSNPEQTAGEERGDGNSGFNEHLKENKVADPVDSSNLDTCGSISQVIEQLPQPN RTSSVLGMSVESAPAVEEEKGEELEQKEKEKEEDTSGNTTHSLGAEDTASSQLGFGVLEL SQSQDVEENTVPYEVDKEQLQSVTTNSGYTRLSDVDANTAIKHEEQSNEDIPIAEQSSKD IPVTAQPSKDVHVVKEQNPPPARSEDMPFSPKASVAAMEAKEQLSAQELMESGLQIQKSP EPEVLSTQEDLFDQSNKTVSSDGCSTPSREEGGCSLASTPATTLHLLQLSGQRSLVQDSL STNSSDLVAPSPDAFRSTPFIVPSSPTEQEGRQDKPMDTSVLSEEGGEPFQKKLQSGEPV ELENPPLLPESTVSPQASTPISQSTPVFPPGSLPIPSQPQFSHDIFIPSPSLEEQSNDGK KDGDMHSSSLTVECSKTSEIEPKNSPEDLGLSLTGDSCKLMLSTSEYSQSPKMESLSSHR IDEDGENTQIEDTEPMSPVLNSKFVPAENDSILMNPAQDGEVQLSQNDDKTKGDDTDTRD DISILATGCKGREETVAEDVCIDLTCDSGSQAVPSPATRSEALSSVLDQEEAMEIKEHHP EEGSSGSEVEEIPETPCESQGEELKEENMESVPLHLSLTETQSQGLCLQKEMPKKECSEA MEVETSVISIDSPQKLAILDQELEHKEQEAWEEATSEDSSVVIVDVKEPSPRVDVSCEPL EGVEKCSDSQSWEDIAPEIEPCAENRLDTKEEKSVEYEGDLKSGTAETEPVEQDSSQPSL PLVRADDPLRLDQELQQPQTQEKTSNSLTEDSKMANAKQLSSDAEAQKLGKPSAHASQSF CESSSETPFHFTLPKEGDIIPPLTGATPPLIGHLKLEPKRHSTPIGISNYPESTIATSDV MSESMVETHDPILGSGKGDSGAAPDVDDKLCLRMKLVSPETEASEESLQFNLEKPATGER KNGSTAVAESVASPQKTMSVLSCICEARQENEARSEDPPTTPIRGNLLHFPSSQGEEEKE KLEGDHTIRQSQQPMKPISPVKDPVSPASQKMVIQGPSSPQGEAMVTDVLEDQKEGRSTN KENPSKALIERPSQNNIGIQTMECSLRVPETVSAATQTIKNVCEQGTSTVDQNFGKQDAT VQTERGSGEKPVSAPGDDTESLHSQGEEEFDMPQPPHGHVLHRHMRTIREVRTLVTRVIT DVYYVDGTEVERKVTEETEEPIVECQECETEVSPSQTGGSSGDLGDISSFSSKASSLHRT SSGTSLSAMHSSGSSGKGAGPLRGKTSGTEPADFALPSSRGGPGKLSPRKGVSQTGTPVC EEDGDAGLGIRQGGKAPVTPRGRGRRGRPPSRTTGTRETAVPGPLGIEDISPNLSPDDKS FSRVVPRVPDSTRRTDVGAGALRRSDSPEIPFQAAAGPSDGLDASSPGNSFVGLRVVAKW SSNGYFYSGKITRDVGAGKYKLLFDDGYECDVLGKDILLCDPIPLDTEVTALSEDEYFSA GVVKGHRKESGELYYSIEKEGQRKWYKRMAVILSLEQGNRLREQYGLGPYEAVTPLTKAA DISLDNLVEGKRKRRSNVSSPATPTASSSSSTTPTRKITESPRASMGVLSGKRKLITSEE ERSPAKRGRKSATVKPGAVGAGEFVSPCESGDNTGEPSALEEQRGPLPLNKTLFLGYAFL LTMATTSDKLASRSKLPDGPTGSSEEEEEFLEIPPFNKQYTESQLRAGAGYILEDFNEAQ CNTAYQCLLIADQHCRTRKYFLCLASGIPCVSHVWVHDSCHANQLQNYRNYLLPAGYSLE EQRILDWQPRENPFQNLKVLLVSDQQQNFLELWSEILMTGGAASVKQHHSSAHNKDIALG VFDVVVTDPSCPASVLKCAEALQLPVVSQEWVIQCLIVGERIGFKQHPKYKHDYVSH >sp|Q12888-3|TP53B_HUMAN Isoform 3 of Tumor suppressor p53- binding protein 1 OS = Homo sapiens GN = TP53BP1 (SEQ ID NO: 285) MPGEQMDPTGSQLDSDFSQQDTPCLIIEDSQPESQVLEDDSGSHFSMLSRHLPNLQTHKE NPVLDVVSNPEQTAGEERGDGNSGFNEHLKENKVADPVDSSNLDTCGSISQVIEQLPQPN RTSSVLGMSVESAPAVEEEKGEELEQKEKEKEEDTSGNTTHSLGAEDTASSQLGFGVLEL SQSQDVEENTVPYEVDKEQLQSVTTNSGYTRLSDVDANTAIKHEEQSNEDIPIAEQSSKD IPVTAQPSKDVHVVKEQNPPPARSEDMPFSPKASVAAMEAKEQLSAQELMESGLQIQKSP EPEVLSTQEDLFDQSNKTVSSDGCSTPSREEGGCSLASTPATTLHLLQLSGQRSLVQDSL STNSSDLVAPSPDAFRSTPFIVPSSPTEQEGRQDKPMDTSVLSEEGGEPFQKKLQSGEPV ELENPPLLPESTVSPQASTPISQSTPVFPPGSLPIPSQPQFSHDIFIPSPSLEEQSNDGK KDGDMHSSSLTVECSKTSEIEPKNSPEDLGLSLTGDSCKLMLSTSEYSQSPKMESLSSHR IDEDGENTQIEDTEPMSPVLNSKFVPAENDSILMNPAQDGEVQLSQNDDKTKGDDTDTRD DISILATGCKGREETVAEDVCIDLTCDSGSQAVPSPATRSEALSSVLDQEEAMEIKEHHP EEGSSGSEVEEIPETPCESQGEELKEENMESVPLHLSLTETQSQGLCLQKEMPKKECSEA MEVETSVISIDSPQKLAILDQELEHKEQEAWEEATSEDSSVVIVDVKEPSPRVDVSCEPL EGVEKCSDSQSWEDIAPEIEPCAENRLDTKEEKSVEYEGDLKSGTAETEPVEQDSSQPSL PLVRADDPLRLDQELQQPQTQEKTSNSLTEDSKMANAKQLSSDAEAQKLGKPSAHASQSF CESSSETPFHFTLPKEGDIIPPLTGATPPLIGHLKLEPKRHSTPIGISNYPESTIATSDV MSESMVETHDPILGSGKGDSGAAPDVDDKLCLRMKLVSPETEASEESLQFNLEKPATGER KNGSTAVAESVASPQKTMSVLSCICEARQENEARSEDPPTTPIRGNLLHFPSSQGEEEKE KLEGDHTIRQSQQPMKPISPVKDPVSPASQKMVIQGPSSPQGEAMVTDVLEDQKEGRSTN KENPSKALIERPSQNNIGIQTMECSLRVPETVSAATQTIKNVCEQGTSTVDQNFGKQDAT VQTERGSGEKPVSAPGDDTESLHSQGEEEFDMPQPPHGHVLHRHMRTIREVRTLVTRVIT DVYYVDGTEVERKVTEETEEPIVECQECETEVSPSQTGGSSGDLGDISSFSSKASSLHRT SSGTSLSAMHSSGSSGKGAGPLRGKTSGTEPADFALPSSRGGPGKLSPRKGVSQTGTPVC EEDGDAGLGIRQGGKAPVTPRGRGRRGRPPSRTTGTRETAVPGPLGIEDISPNLSPDDKS FSRVVPRVPDSTRRTDVGAGALRRSDSPEIPFQAAAGPSDGLDASSPGNSFVGLRVVAKW SSNGYFYSGKITRDVGAGKYKLLFDDGYECDVLGKDILLCDPIPLDTEVTALSEDEYFSA GVVKGHRKESGELYYSIEKEGQRKWYKRMAVILSLEQGNRLREQYGLGPYEAVTPLTKAA DISLDNLVEGKRKRRSNVSSPATPTASSSSSTTPTRKITESPRASMGVLSGKRKLITSEE ERSPAKRGRKSATVKPVGAGEFVSPCESGDNTGEPSALEEQRGPLPLNKTLFLGYAFLLT MATTSDKLASRSKLPDGPTGSSEEEEEFLEIPPFNKQYTESQLRAGAGYILEDFNEAQCN TAYQCLLIADQHCRTRKYFLCLASGIPCVSHVWVHDSCHANQLQNYRNYLLPAGYSLEEQ RILDWQPRENPFQNLKVLLVSDQQQNFLELWSEILMTGGAASVKQHHSSAHNKDIALGVF DVVVTDPSCPASVLKCAEALQLPVVSQEWVIQCLIVGERIGFKQHPKYKHDYVSH XRCC4 >sp|Q13426|XRCC4_HUMAN DNA repair protein XRCC4 OS = Homo sapiens GN = XRCC4 PE = 1 SV = 2 (SEQ ID NO: 286) MERKISRIHLVSEPSITHFLQVSWEKTLESGFVITLTDGHSAWTGTVSESEISQEADDMA MEKGKYVGELRKALLSGAGPADVYTFNFSKESCYFFFEKNLKDVSFRLGSFNLEKVENPA EVIRELICYCLDTIAENQAKNEHLQKENERLLRDWNDVQGRFEKCVSAKEALETDLYKRF ILVLNEKKTKIRSLHNKLLNAAQEREKDIKQEGETAICSEMTADRDPVYDESTDEESENQ TDLSGLASAAVSKDDSIISSLDVTDIAPSRKRRQRMQRNLGTEPKMAPQENQLQEKENSR PDSSLPETSKKEHISAENMSLETLRNSSPEDLFDEI >sp|Q13426-2|XRCC4_HUMAN Isoform 2 of DNA repair protein XRCC4 OS = Homo sapiens GN = XRCC4 (SEQ ID NO: 287) MERKISRIHLVSEPSITHFLQVSWEKTLESGFVITLTDGHSAWTGTVSESEISQEADDMA MEKGKYVGELRKALLSGAGPADVYTFNFSKESCYFFFEKNLKDVSFRLGSFNLEKVENPA EVIRELICYCLDTIAENQAKNEHLQKENERLLRDWNDVQGRFEKCVSAKEALETDLYKRF ILVLNEKKTKIRSLHNKLLNAAQEREKDIKQEGETAICSEMTADRDPVYDESTDEESENQ TDLSGLASAAVSKDDSIISSLDVTDIAPSRKRRQRMQRNLGTEPKMAPQENQLQEKEKPD SSLPETSKKEHISAENMSLETLRNSSPEDLFDEI >sp|Q13426-3|XRCC4_HUMAN Isoform 3 of DNA repair protein XRCC4 OS = Homo sapiens GN = XRCC4 (SEQ ID NO: 288) MERKISRIHLVSEPSITHFLQVSWEKTLESGFVITLTDGHSAWTGTVSESEISQEADDMA MEKGKYVGELRKALLSGAGPADVYTFNFSKESCYFFFEKNLKDVSFRLGSFNLEKVENPA EVIRELICYCLDTIAENQAKNEHLQKENERLLRDWNDVQGRFEKCVSAKEALETDLYKRF ILVLNEKKTKIRSLHNKLLNAAQEREKDIKQEGETAICSEMTADRDPVYDESTDEESENQ TDLSGLASAAVSKDDSIISSLDVTDIAPSRKRRQRMQRNLGTEPKMAPQENQLQEKEKGR KKETSEKEAV LIG4 >sp|P49917|DNLI4_HUMAN DNA ligase 4 OS = Homo sapiens GN = LIG4 PE = 1 SV = 2 (SEQ ID NO: 289) MAASQTSQTVASHVPFADLCSTLERIQKSKGRAEKIRHFREFLDSWRKFHDALHKNHKDV TDSFYPAMRLILPQLERERMAYGIKETMLAKLYIELLNLPRDGKDALKLLNYRIPTGTHG DAGDFAMIAYFVLKPRCLQKGSLTIQQVNDLLDSIASNNSAKRKDLIKKSLLQLITQSSA LEQKWLIRMIIKDLKLGVSQQTIFSVFHNDAAELHNVTTDLEKVCRQLHDPSVGLSDISI TLFSAFKPMLAAIADIEHIEKDMKHQSFYIETKLDGERMQMHKDGDVYKYFSRNGYNYTD QFGASPTEGSLIPFIHNAFKADIQICILDGEMMAYNPNIQTFMQKGTKFDIKRMVEDSDL QTCYCVFDVLMVNNKKLGHETLRKRYEILSSIFTPIPGRIEIVQKTQAHTKNEVIDALNE AIDKREEGIMVKQPLSIYKPDKRGEGWLKIKPEYVSGLMDELDILIVGGYWGKGSRGGMM SHFLCAVAEKPPPGEKPSVFHTLSRVGSGCTMKELYDLGLKLAKYWKPFHRKAPPSSILC GTEKPEVYIEPCNSVIVQIKAAEIVPSDMYKTGCTLRFPRIEKIRDDKEWHECMTLDDLE QLRGKASGKLASKHLYIGGDDEPQEKKRKAAPKMKKVIGIIEHLKAPNLTNVNKISNIFE DVEFCVMSGTDSQPKPDLENRIAEFGGYIVQNPGPDTYCVIAGSENIRVKNIILSNKHDV VKPAWLLECFKTKSFVPWQPRFMIHMCPSTKEHFAREYDCYGDSYFIDTDLNQLKEVFSG IKNSNEQTPEEMASLIADLEYRYSWDCSPLSMFRRHTVYLDSYAVINDLSTKNEGTRLAI KALELRFHGAKVVSCLAEGVSHVIIGEDHSRVADFKAFRRTFKRKFKILKESWVTDSIDK CELQEENQYLI XLF (also >sp|Q9H9Q4|NHEJ1_HUMAN Non-homologous end-joining factor 1 called NHEJ1) OS = Homo sapiens GN = NHEJ1 PE = 1 SV = 1 (SEQ ID NO: 290) MEELEQGLLMQPWAWLQLAENSLLAKVFITKQGYALLVSDLQQVWHEQVDTSVVSQRAKE LNKRLTAPPAAFLCHLDNLLRPLLKDAAHPSEATFSCDCVADALILRVRSELSGLPFYWN FHCMLASPSLVSQHLIRPLMGMSLALQCQVRELATLLHMKDLEIQDYQESGATLIRDRLK TEPFEENSFLEQFMIEKLPEACSIGDGKPFVMNLQDLYMAVTTQEVQVGQKHQGAGDPHT SNSASLQGIDSQCVNQPEQLVSSAPTLSAPEKESTGTSGPLQRPQLSKVKRKKPRGLFS >sp|Q9H9Q4-2|NHEJ1_HUMAN Isoform 2 of Non-homologous end-joining factor 1 OS = Homo sapiens GN = NHEJ1 (SEQ ID NO: 291) MEELEQGLLMQPWAWLQLAENSLLAKVFITKQGYALLVSDLQQVWHEQVDTSVVSQRAKE LNKRLTAPPAAFLCHLDNLLRPLLKDAAHPSEATFSCDCVADALILRVRSELSGLPFYWN FHCMLASPSLVSQHLIRPLMGMSLALQCQVRELATLLHMKDLEIQDYQESGATLIRDRLK TEPFEENSFLEQFMIEKLPEACSIGDGKPFVMNLQDLYMAVTTQEVQVGQKHQGAGDPHT SNSASLQGIDSQCVNQPEQLVSSAPTLSAPEKESTALCRDLSCQRSRGRSQGVSSVNLLW PQLLRMDLENSFQASP ARTEMIS MSSFEGQMAEYPTISIDRFDRENLRARAYFLSHCHKDHMKGLRAPTLKRRLECSLKVYLY CSPVTKELLLTSPKYRFWKKRIISIEIETPTQISLVDEASGEKEEIVVTLLPAGHCPGSV MFLFQGNNGTVLYTGDFRLAQGEAARMELLHSGGRVKDIQSVYLDTTFCDPRFYQIPSRE ECLSGVLELVRSWITRSPYHVVWLNCKAAYGYEYLFTNLSEELGVQVHVNKLDMFRNMPE ILHHLTTDRNTQIHACRHPKAEEYFQWSKLPCGITSRNRIPLHIISIKPSTMWFGERSRK TNVIVRTGESSYRACFSFHSSYSEIKDFLSYLCPVNAYPNVIPVGTTMDKVVEILKPLCR SSQSTEPKYKPLGKLKRARTVHRDSEEEDDYLFDDPLPIPLRHKVPYPETFHPEVFSMTA VSEKQPEKLRQTPGCCRAECMQSSRFTNFVDCEESNSESEEEVGIPASLQGDLGSVLHLQ KADGDVPQWEVFFKRNDEITDESLENFPSSTVAGGSQSPKLFSDSDGESTHISSQNSSQS THITEQGSQGWDSQSDTVLLSSQERNSGDITSLDKADYRPTIKENIPASLMEQNVICPKD TYSDLKSRDKDVTIVPSTGEPTTLSSETHIPEEKSLLNLSTNADSQSSSDFEVPSTPEAE LPKREHLQYLYEKLATGESIAVKKRKCSLLDT (Artemis Isoform 1 CCDS 31149.1) (SEQ ID NO: 292) MKHQERFLFQGNNGTVLYTGDFRLAQGEAARMELLHSGGRVKDIQSVYLDTTFCDPRFYQ IPSREECLSGVLELVRSWITRSPYHVVWLNCKAAYGYEYLFTNLSEELGVQVHVNKLDMF RNMPEILHHLTTDRNTQIHACRHPKAEEYFQWSKLPCGITSRNRIPLHIISIKPSTMWFG ERSRKTNVIVRTGESSYRACFSFHSSYSEIKDFLSYLCPVNAYPNVIPVGTTMDKVVEIL KPLCRSSQSTEPKYKPLGKLKRARTVHRDSEEEDDYLFDDPLPIPLRHKVPYPETFHPEV FSMTAVSEKQPEKLRQTPGCCRAECMQSSRFTNFVDCEESNSESEEEVGIPASLQGDLGS VLHLQKADGDVPQWEVFFKRNDEITDESLENFPSSTVAGGSQSPKLFSDSDGESTHISSQ NSSQSTHITEQGSQGWDSQSDTVLLSSQERNSGDITSLDKADYRPTIKENIPASLMEQNV ICPKDTYSDLKSRDKDVTIVPSTGEPTTLSSETHIPEEKSLLNLSTNADSQSSSDFEVPS TPEAELPKREHLQYLYEKLATGESIAVKKRKCSLLDT (Artemis Isoform 2 CCDS 7105.1) (SEQ ID NO: 293) MFLFQGNNGTVLYTGDFRLAQGEAARMELLHSGGRVKDIQSVYLDTTFCDPRFYQIPSRE ECLSGVLELVRSWITRSPYHVVWLNCKAAYGYEYLFTNLSEELGVQVHVNKLDMFRNMPE ILHHLTTDRNTQIHACRHPKAEEYFQWSKLPCGITSRNRIPLHIISIKPSTMWFGERSRK TNVIVRTGESSYRACFSFHSSYSEIKDFLSYLCPVNAYPNVIPVGTTMDKVVEILKPLCR SSQSTEPKYKPLGKLKRARTVHRDSEEEDDYLFDDPLPIPLRHKVPYPETFHPEVFSMTA VSEKQPEKLRQTPGCCRAECMQSSRFTNFVDCEESNSESEEEVGIPASLQGDLGSVLHLQ KADGDVPQWEVFFKRNDEITDESLENFPSSTVAGGSQSPKLFSDSDGESTHISSQNSSQS THITEQGSQGWDSQSDTVLLSSQERNSGDITSLDKADYRPTIKENIPASLMEQNVICPKD TYSDLKSRDKDVTIVPSTGEPTTLSSETHIPEEKSLLNLSTNADSQSSSDFEVPSTPEAE LPKREHLQYLYEKLATGESIAVKKRKCSLLDT (Artemis Isoform 3 CCDS 31150.1) (SEQ ID NO: 294) APLF >sp|Q8IW19|APLF_HUMAN Aprataxin and PNK-like factor OS = Homo sapiens GN = APLF PE = 1 SV = 1 (SEQ ID NO: 295) MSGGFELQPRDGGPRVALAPGETVIGRGPLLGITDKRVSRRHAILEVAGGQLRIKPIHTN PCFYQSSEKSQLLPLKPNLWCYLNPGDSFSLLVDKYIFRILSIPSEVEMQCTLRNSQVLD EDNILNETPKSPVINLPHETTGASQLEGSTEIAKTQMTPTNSVSFLGENRDCNKQQPILA ERKRILPTWMLAEHLSDQNLSVPAISGGNVIQGSGKEEICKDKSQLNTTQQGRRQLISSG SSENTSAEQDTGEECKNTDQEESTISSKEMPQSFSAITLSNTEMNNIKTNAQRNKLPIEE LGKVSKHKIATKRIPHKEDEAMSCSENCSSAQGDSLQDESQGSHSESSSNPSNPETLHAK ATDSVLQGSEGNKVKRTSCMYGANCYRKNPVHFQHFSHPGDSDYGGVQIVGQDETDDRPE CPYGPSCYRKNPQHKIEYRHNTLPVRNVLDEDNDNVGQPNEYDLNDSFLDDEEEDYEPTD EDSDWEPGKEDEEKEDVEELLKEAKRFMKRK PNK (also >sp|Q96T60|PNKP_HUMAN Bifunctional polynucleotide called PNKP) phosphatase/kinase OS = Homo sapiens GN = PNKP PE = 1 SV = 1 (SEQ ID NO: 296) MGEVEAPGRLWLESPPGGAPPIFLPSDGQALVLGRGPLTQVTDRKCSRTQVELVADPETR TVAVKQLGVNPSTTGTQELKPGLEGSLGVGDTLYLVNGLHPLTLRWEETRTPESQPDTPP GTPLVSQDEKRDAELPKKRMRKSNPGWENLEKLLVFTAAGVKPQGKVAGFDLDGTLITTR SGKVFPTGPSDWRILYPEIPRKLRELEAEGYKLVIFTNQMSIGRGKLPAEEFKAKVEAVV EKLGVPFQVLVATHAGLYRKPVTGMWDHLQEQANDGTPISIGDSIFVGDAAGRPANWAPG RKKKDFSCADRLFALNLGLPFATPEEFFLKWPAAGFELPAFDPRTVSRSGPLCLPESRAL LSASPEVVVAVGFPGAGKSTFLKKHLVSAGYVHVNRDTLGSWQRCVTTCETALKQGKRVA IDNTNPDAASRARYVQCARAAGVPCRCFLFTATLEQARHNNRFREMTDSSHIPVSDMVMY GYRKQFEAPTLAEGFSAILEIPFRLWVEPRLGRLYCQFSEG >sp|Q96T60-2|PNKP_HUMAN Isoform 2 of Bifunctional polynucleotide phosphatase/kinase OS = Homo sapiens GN = PNKP (SEQ ID NO: 297) MQILTPPLQSSVELVADPETRTVAVKQLGVNPSTTGTQELKPGLEGSLGVGDTLYLVNGL HPLTLRWEETRTPESQPDTPPGTPLVSQDEKRDAELPKKRMRKSNPGWENLEKLLVFTAA GVKPQGKVAGFDLDGTLITTRSGKVFPTGPSDWRILYPEIPRKLRELEAEGYKLVIFTNQ MSIGRGKLPAEEFKAKVEAVVEKLGVPFQVLVATHAGLYRKPVTGMWDHLQEQANDGTPI SIGDSIFVGDAAGRPANWAPGRKKKDFSCADRLFALNLGLPFATPEEFFLKWPAAGFELP AFDPRIVSRSGPLCLPESRALLSASPEVVVAVGFPGAGKSTFLKKHLVSAGYVHVNRDIL GSWQRCVTTCETALKQGKRVAIDNTNPDAASRARYVQCARAAGVPCRCFLFTATLEQARH NNRFREMIDSSHIPVSDMVMYGYRKQFEAPTLAEGFSAILEIPFRLWVEPRLGRLYCQFS EG Rif1 >sp|Q5UIP0|RIF1_HUMAN Telomere-associated protein RIF1 OS = Homo sapiens GN = RIF1 PE = 1 SV = 2 (SEQ ID NO: 298) MTARGQSPLAPLLETLEDPSASHGGQTDAYLTLTSRMTGEEGKEVITEIEKKLPRLYKVL KTHISSQNSELSSAALQALGFCLYNPKITSELSEANALELLSKLNDTIKNSDKNVRTRAL WVISKQTFPSEVVGKMVSSIIDSLEILFNKGETHSAVVDFEALNVIVRLIEQAPIQMGEE AVRWAKLVIPLVVHSAQKVHLRGATALEMGMPLLLQKQQEIASITEQLMTTKLISELQKL FMSKNETYVLKLWPLFVKLLGRTLHRSGSFINSLLQLEELGFRSGAPMIKKIAFIAWKSL IDNFALNPDILCSAKRLKLLMQPLSSIHVRTETLALTKLEVWWYLLMRLGPHLPANFEQV CVPLIQSTISIDSNASPQGNSCHVATSPGLNPMTPVHKGASSPYGAPGTPRMNLSSNLGG MATIPSIQLLGLEMLLHFLLGPEALSFAKQNKLVLSLEPLEHPLISSPSFFSKHANTLIT AVHDSFVAVGKDAPDVVVSAIWKELISLVKSVTESGNKKEKPGSEVLTLLLKSLESIVKS EVFPVSKTLVLMEITIKGLPQKVLGSPAYQVANMDILNGTPALFLIQLIFNNFLECGVSD ERFFLSLESLVGCVLSGPTSPLAFSDSVLNVINQNAKQLENKEHLWKMWSVIVTPLTELI NQTNEVNQGDALEHNFSAIYGALTLPVNHIFSEQRFPVATMKTLLRTWSELYRAFARCAA LVATAEENLCCEELSSKIMSSLEDEGFSNLLFVDRIIYIITVMVDCIDFSPYNIKYQPKV KSPQRPSDWSKKKNEPLGKLTSLFKLIVKVIYSFHTLSFKEAHSDTLFTIGNSITGIISS VLGHISLPSMIRKIFATLTRPLALFYENSKLDEVPKVYSCLNNKLEKLLGETIACLQFSY TGTYDSELLEQLSPLLCIIFLHKNKQIRKQSAQFWNATFAKVMMLVYPEELKPVLTQAKQ KFLLLLPGLETVEMMEESSGPYSDGTENSQLNVKISGMERKSNGKRDSFLAQTKNKKENM KPAAKLKLESSSLKVKGEILLEEEKSTDFVFIPPEGKDAKERILTDHQKEVLKTKRCDIP AMYNNLDVSQDTLFTQYSQEEPMEIPTLTRKPKEDSKMMITEEQMDSDIVIPQDVTEDCG MAEHLEKSSLSNNECGSLDKISPEMSNSNNDERKKALISSRKTSTECASSTENSFVVSSS SVSNTTVAGTPPYPTSRRQTFITLEKFDGSENRPFSPSPLNNISSTVTVKNNQETMIKTD FLPKAKQREGTFSKSDSEKIVNGTKRSSRRAGKAEQTGNKRSKPLMRSEPEKNTEESVEG IVVLENNPPGLLNQTECVSDNQVHLSESTMEHDNTKLKAATVENAVLLETNTVEEKNVEI NLESKENTPPVVISADQMVNEDSQVQIIPNQKTLRRSSRRRSEVVESTTESQDKENSHQK KERRKEEEKPLQKSPLHIKDDVLPKQKLIAEQTLQENLIEKGSNLHEKTLGETSANAETE QNKKKADPENIKSEGDGTQDIVDKSSEKLVRGRTRYQTRRASQGLLSSIENSESDSSEAK EEGSRKKRSGKWKNKSNESVDIQDQEEKVVKQECIKAENQSHDYKATSEEDVSIKSPICE KQDESNTVICQDSTVTSDLLQVPDDLPNVCEEKNETSKYAEYSFTSLPVPESNLRTRNAI KRLHKRDSFDNCSLGESSKIGISDISSLSEKTFQTLECQHKRSRRVRRSKGCDCCGEKSQ PQEKSLIGLKNTENNDVEISETKKADVQAPVSPSETSQANPYSEGQFLDEHHSVNFHLGL KEDNDTINDSLIVSETKSKENTMQESLPSGIVNFREEICDMDSSEAMSLESQESPNENFK TVGPCLGDSKNVSQESLETKEEKPEETPKMELSLENVTVEGNACKVTESNLEKAKTMELN VGNEASFHGQERTKTGISEEAAIEENKRNDDSEADTAKLNAKEVATEEFNSDISLSDNTT PVKLNAQTEISEQTAAGELDGGNDVSDLHSSEETNIKMKNNEEMMIGEAMAEIGHDGETE NEGITTKTSKPDEAETNMLTAEMDNFVCDTVEMSTEEGIIDANKTETNTEYSKSEEKLDN NQMVMESDILQEDHHTSQKVEEPSQCLASGTAISELIIEDNNASPQKLRELDPSLVSAND SPSGMQTRCVWSPLASPSTSILKRGLKRSQEDEISSPVNKVRRVSFADPIYQAGLADDID RRCSIVRSHSSNSSPIGKSVKISPITQSKHNITSAKGFLSPGSRSPKFKSSKKCLISEMA KESIPCPTESVYPPLVNCVAPVDIILPQITSNMWARGLGQLIRAKNIKTIGDLSTLTASE IKTLPIRSPKVSNVKKALRIYHEQQVKTRGLEEIPVFDISEKTVNGIENKSLSPDEERLV SDIIDPVALEIPLSKNLLAQISALALQLDSEDLHNYSGSQLFEMHEKLSCMANSVIKNLQ SRWRSPSHENSI >sp|Q5UIP0-2|RIF1_HUMAN Isoform 2 of Telomere-associated protein RIF1 OS = Homo sapiens GN = RIF1 (SEQ ID NO: 299) MTARGQSPLAPLLETLEDPSASHGGQTDAYLTLTSRMTGEEGKEVITEIEKKLPRLYKVL KTHISSQNSELSSAALQALGFCLYNPKITSELSEANALELLSKLNDTIKNSDKNVRTRAL WVISKQTEPSEVVGKMVSSIIDSLEILFNKGETHSAVVDFEALNVIVRLIEQAPIQMGEE AVRWAKLVIPLVVHSAQKVHLRGATALEMGMPLLLQKQQEIASITEQLMTTKLISELQKL FMSKNETYVLKLWPLFVKLLGRTLHRSGSFINSLLQLEELGFRSGAPMIKKIAFIAWKSL IDNFALNPDILCSAKRLKLLMQPLSSIHVRTETLALTKLEVWWYLLMRLGPHLPANFEQV CVPLIQSTISIDSNASPQGNSCHVATSPGLNPMTPVHKGASSPYGAPGTPRMNLSSNLGG MATIPSIQLLGLEMLLHFLLGPEALSFAKQNKLVLSLEPLEHPLISSPSFFSKHANTLIT AVHDSFVAVGKDAPDVVVSAIWKELISLVKSVTESGNKKEKPGSEVLTLLLKSLESIVKS EVFPVSKTLVLMEITIKGLPQKVLGSPAYQVANMDILNGTPALFLIQLIFNNFLECGVSD ERFFLSLESLVGCVLSGPTSPLAFSDSVLNVINQNAKQLENKEHLWKMWSVIVTPLTELI NQTNEVNQGDALEHNFSAIYGALTLPVNHIFSEQRFPVATMKTLLRTWSELYRAFARCAA LVATAEENLCCEELSSKIMSSLEDEGFSNLLFVDRIIYIITVMVDCIDFSPYNIKYQPKV KSPQRPSDWSKKKNEPLGKLTSLFKLIVKVIYSFHTLSFKEAHSDTLFTIGNSITGIISS VLGHISLPSMIRKIFATLTRPLALFYENSKLDEVPKVYSCLNNKLEKLLGETIACLQFSY TGTYDSELLEQLSPLLCIIFLHKNKQIRKQSAQFWNATFAKVMMLVYPEELKPVLTQAKQ KFLLLLPGLETVEMMEESSGPYSDGTENSQLNVKISGMERKSNGKRDSFLAQTKNKKENM KPAAKLKLESSSLKVKGEILLEEEKSTDFVFIPPEGKDAKERILTDHQKEVLKTKRCDIP AMYNNLDVSQDTLFTQYSQEEPMEIPTLTRKPKEDSKMMITEEQMDSDIVIPQDVTEDCG MAEHLEKSSLSNNECGSLDKTSPEMSNSNNDERKKALISSRKTSTECASSTENSFVVSSS SVSNTTVAGTPPYPTSRRQTFITLEKFDGSENRPFSPSPLNNISSTVTVKNNQETMIKTD FLPKAKQREGTFSKSDSEKIVNGTKRSSRRAGKAEQTGNKRSKPLMRSEPEKNTEESVEG IVVLENNPPGLLNQTECVSDNQVHLSESTMEHDNTKLKAATVENAVLLETNTVEEKNVEI NLESKENTPPVVISADQMVNEDSQVQITPNQKTLRRSSRRRSEVVESTTESQDKENSHQK KERRKEEEKPLQKSPLHIKDDVLPKQKLIAEQTLQENLIEKGSNLHEKTLGETSANAETE QNKKKADPENIKSEGDGTQDIVDKSSEKLVRGRTRYQTRRASQGLLSSIENSESDSSEAK EEGSRKKRSGKWKNKSNESVDIQDQEEKVVKQECIKAENQSHDYKATSEEDVSIKSPICE KQDESNTVICQDSTVTSDLLQVPDDLPNVCEEKNETSKYAEYSFTSLPVPESNLRTRNAI KRLHKRDSFDNCSLGESSKIGISDISSLSEKTFQTLECQHKRSRRVRRSKGCDCCGEKSQ PQEKSLIGLKNTENNDVEISETKKADVQAPVSPSETSQANPYSEGQFLDEHHSVNFHLGL KEDNDTINDSLIVSETKSKENTMQESLPSGIVNFREEICDMDSSEAMSLESQESPNENFK TVGPCLGDSKNVSQESLETKEEKPEETPKMELSLENVTVEGNACKVTESNLEKAKTMELN VGNEASFHGQERTKTGISEEAAIEENKRNDDSEADTAKLNAKEVATEEFNSDISLSDNTT PVKLNAQTEISEQTAAGELDGGNDVSDLHSSEETNTKMKNNEEMMIGEAMAETGHDGETE NEGITTKTSKPDEAETNMLTAEMDNFVCDTVEMSTEEGIIDANKTETNTEYSKSEEKLDN NQMVMESDILQEDHHTSQKVEEPSQCLASGTAISELIIEDNNASPQKLRELDPSLVSAND SPSGMQTRCVWSPLASPSTSILKRGLKRSQEDEISSPVNKVRRVSFADPIYQAGLADDID RRCSIVRSHSSNSSPIGKSVKTSPTTQSKISEMAKESIPCPTESVYPPLVNCVAPVDIIL PQITSNMWARGLGQLIRAKNIKTIGDLSTLTASEIKTLPIRSPKVSNVKKALRIYHEQQV KTRGLEEIPVFDISEKTVNGIENKSLSPDEERLVSDIIDPVALEIPLSKNLLAQISALAL QLDSEDLHNYSGSQLFEMHEKLSCMANSVIKNLQSRWRSPSHENSI PTIP (also >sp|Q6ZW49|PAXI1_HUMAN PAX-interacting protein 1 OS = Homo sapiens  called PAXIP1) GN = PAXIP1 PE = 1 SV = 2 (SEQ ID NO: 300) MSDQAPKVPEEMFREVKYYAVGDIDPQVIQLLKAGKAKEVSYNALASHIISEDGDNPEVG EAREVFDLPVVKPSWVILSVQCGTLLPVNGFSPESCQIFFGITACLSQVSSEDRSALWAL VTFYGGDCQLTLNKKCTHLIVPEPKGEKYECALKRASIKIVTPDWVLDCVSEKTKKDEAF YHPRLIIYEEEEEEEEEEEEVENEEQDSQNEGSTDEKSSPASSQEGSPSGDQQFSPKSNT EKSKGELMFDDSSDSSPEKQERNLNWTPAEVPQLAAAKRRLPQGKEPGLINLCANVPPVP GNILPPEVRGNLMAAGQNLQSSERSEMIATWSPAVRTLRNITNNADIQQMNRPSNVAHIL QTLSAPTKNLEQQVNHSQQGHTNANAVLFSQVKVTPETHMLQQQQQAQQQQQQHPVLHLQ PQQIMQLQQQQQQQISQQPYPQQPPHPFSQQQQQQQQAHPHQFSQQQLQFPQQQLHPPQQ LHRPQQQLQPFQQQHALQQQFHQLQQHQLQQQQLAQLQQQHSLLQQQQQQQIQQQQLQRM HQQQQQQQMQSQTAPHLSQTSQALQHQVPPQQPPQQQQQQQPPPSPQQHQLFGHDPAVEI PEEGFLLGCVFAIADYPEQMSDKQLLATWKRITQAHGGTVDPTFTSRCTHLLCESQVSSA YAQAIRERKRCVTAHWLNTVLKKKKMVPPHRALHFPVAFPPGGKPCSQHIISVTGFVDSD RDDLKLMAYLAGAKYTGYLCRSNTVLICKEPTGLKYEKAKEWRIPCVNAQWLGDILLGNF EALRQIQYSRYTAFSLQDPFAPTQHLVLNLLDAWRVPLKVSAELLMSIRLPPKLKQNEVA NVQPSSKRARIEDVPPPTKKLTPELTPFVLFTGFEPVQVQQYIKKLYILGGEVAESAQKC THLIASKVTRTVKFLTAISVVKHIVTPEWLEECFRCQKFIDEQNYILRDAEAEVLFSFSL EESLKRAHVSPLFKAKYFYITPGICPSLSTMKAIVECAGGKVLSKQPSFRKLMEHKQNSS LSEIILISCENDLHLCREYFARGIDVHNAEFVLTGVLTQTLDYESYKFN >sp|Q6ZW49-2|PAXI1_HUMAN Isoform 3 of PAX-interacting protein 1 OS = Homo sapiens GN = PAXIP1 (SEQ ID NO: 301) MFDDSSDSSPEKQERNLNWTPAEVPQLAAAKRRLPQGKEPGLINLCANVPPVPGNILPPE VRGNLMAAGQNLQSSERSEMIATWSPAVRTLRNITNNADIQQMNRPSNVAHILQTLSAPT KNLEQQVNHSQQGHTNANAVLFSQVKVTPETHMLQQQQQAQQQQQQHPVLHLQPQQIMQL QQQQQQQISQQPYPQQPPHPFSQQQQQQQQAHPHQFSQQQLQFPQQQLHPPQQLHRPQQQ LQPFQQQHALQQQFHQLQQHQLQQQQLAQLQQQHSLLQQQQQQQIQQQQLQRMHQQQQQQ QMQSQTAPHLSQTSQALQHQVPPQQPPQQQQQQQPPPSPQQHQLFGHDPAVEIPEEGFLL GCVFAIADYPEQMSDKQLLATWKRITQAHGGTVDPTFTSRCTHLLCESQVSSAYAQAIRE RKRCVTAHWLNTVLKKKKMVPPHRALHFPVAFPPGGKPCSQHIISVTGFVDSDRDDLKLM AYLAGAKYTGYLCRSNTVLICKEPTGLKYEKAKEWRIPCVNAQWLGDILLGNFEALRQIQ YSRYTAFSLQDPFAPTQHLVLNLLDAWRVPLKVSAELLMSIRLPPKLKQNEVANVQPSSK RARIEDVPPPTKKLTPELTPFVLFTGFEPVQVQQYIKKLYILGGEVAESAQKCTHLIASK VTRTVKFLTAISVVKHIVTPEWLEECFRCQKFIDEQNYILRDAEAEVLFSFSLEESLKRA HVSPLFKAKYFYITPGICPSLSTMKAIVECAGGKVLSKQPSFRKLMEHKQNSSLSEIILI SCENDLHLCREYFARGIDVHNAEFVLTGVLTQTLDYESYKFN >sp|Q6ZW49|PAXI1_HUMAN PAX-interacting protein 1 OS = Homo sapiens  GN = PAXIP1 PE = 1 SV = 2 (SEQ ID NO: 302) MSDQAPKVPEEMFREVKYYAVGDIDPQVIQLLKAGKAKEVSYNALASHIISEDGDNPEVG EAREVFDLPVVKPSWVILSVQCGTLLPVNGFSPESCQIFFGITACLSQVSSEDRSALWAL VTFYGGDCQLTLNKKCTHLIVPEPKGEKYECALKRASIKIVTPDWVLDCVSEKTKKDEAF YHPRLIIYEEEEEEEEEEEEVENEEQDSQNEGSTDEKSSPASSQEGSPSGDQQFSPKSNT EKSKGELMFDDSSDSSPEKQERNLNWTPAEVPQLAAAKRRLPQGKEPGLINLCANVPPVP GNILPPEVRGNLMAAGQNLQSSERSEMIATWSPAVRTLRNITNNADIQQMNRPSNVAHIL QTLSAPTKNLEQQVNHSQQGHTNANAVLFSQVKVTPETHMLQQQQQAQQQQQQHPVLHLQ PQQIMQLQQQQQQQISQQPYPQQPPHPFSQQQQQQQQAHPHQFSQQQLQFPQQQLHPPQQ LHRPQQQLQPFQQQHALQQQFHQLQQHQLQQQQLAQLQQQHSLLQQQQQQQIQQQQLQRM HQQQQQQQMQSQTAPHLSQTSQALQHQVPPQQPPQQQQQQQPPPSPQQHQLFGHDPAVEI PEEGFLLGCVFAIADYPEQMSDKQLLATWKRITQAHGGTVDPTFTSRCTHLLCESQVSSA YAQAIRERKRCVTAHWLNTVLKKKKMVPPHRALHFPVAFPPGGKPCSQHIISVTGFVDSD RDDLKLMAYLAGAKYTGYLCRSNTVLICKEPTGLKYEKAKEWRIPCVNAQWLGDILLGNF EALRQIQYSRYTAFSLQDPFAPTQHLVLNLLDAWRVPLKVSAELLMSIRLPPKLKQNEVA NVQPSSKRARIEDVPPPTKKLTPELTPFVLFTGFEPVQVQQYIKKLYILGGEVAESAQKC THLIASKVTRTVKFLTAISVVKHIVTPEWLEECFRCQKFIDEQNYILRDAEAEVLFSFSL EESLKRAHVSPLFKAKYFYITPGICPSLSTMKAIVECAGGKVLSKQPSFRKLMEHKQNSS LSEIILISCENDLHLCREYFARGIDVHNAEFVLTGVLTQTLDYESYKFN DNA polymerase —

More specifically, in some embodiments canonical NHEJ is down-regulated using NU7441 (which inhibits mTor and DNA Pk; see, e.g., Robert et al. (2015) GENOME MED. 7(1): 93), KU-0060648 (which also inhibits DNA Pk; see, e.g., Robert et al. (2015)), CC 115 (which inhibits mTor and DNA Pk), NK314 (which inhibits a topoisomerase and DNA Pk), or an siRNA against 53BP1, or an agent of Table VI.8, or any combination thereof.

TABLE VI.8 Down-regulators of the canonical NHEJ pathway. 1. DNA Pk Inhibitors Compounds Phase Target NU7441 mTor and DNAPk KU-0060648 DNAPk CC115 Phase I mTor and DNAPk NK314 Phase I Topo and DNAPk Wortmannin LY294002 NU 7026 IC86621 IC87102 IC87361 OK1035 SU11752 IC486241 Vaillin siRNAs Accell Human PRKDC siRNA, sold by Dharmacon PRKDC Silencer, sold by Life Technologies antibodies DNA-Pk antibody, sold by Biorbyt Anti-DNA-PK (Ab-2) Mouse mAh (18-2), sold by EMD Millipore 2. LigIV Compounds Reference(s) SCR7 See, e.g., Srivastava et al. (2012) CELL 151(7): 1474-1487; and Maruyama et al. (2015) NAT BIOTECHNOL. 33(5): 538-42. siRNA Commercially available from Dharmacon or Ambion 3. 53BP1 siRNAs TP53BP1 Silencer, sold by Life Technologies Accell Human TP53BP1 siRNA, sold by Dharmacon CAGATATCAGCTTAGACAA (SEQ ID NO: 303) antibodies Anti-53BP1, clone BP13, sold by EMD Millipore TP53BP1 monoclonal antibody (M01), clone 1B9, sold by Abnova Corporation 4. Rif1-interacting with 53BP1 siRNA Commercially available from Dharmacon or Ambion 5. PTIP-interacting with 53BP1 siRNA Commercially available from Dharmacon or Ambion

VI.4 Alternative Non-Homologous End-Joining (Alt-NHEJ)

Alt-NHEJ appears to encompass a variety of different DNA repair processes, including blut EJ, MMEJ, and SD-MMEJ (see FIG. 1 ). The common feature is that alt-NHEJ is independent from KU70/80 and Xrcc4/Ligase IV, and is associated with deletion at the repair junctions.

Alternative NHEJ has different subclasses, some of which have specific names like: MMEJ (microhomology mediated end-joining) and SDMMEJ (synthesis dependent micro homology mediated end-joining), and others that do not have specific names but are characterized by not having any microhomology at the break-point. In MMEJ, a limited amount of resection occurs and there is microhomology at the break site (typically 5-25 bp); MMEJ is one of the most abundant and characterized types of alt-NHEJ. In SDMMEJ, there is de novo synthesis by an accurate non-processive DNA polymerase that creates microhomology.

Alt-NHEJ is also mostly independent from DNAPk (a key participant in canonical NHEJ, as discussed above), and is instead dependent on the MRN complex (composed of MRE11, Rad50 and Nbs1) and CtIP, both of which participate in resection.

PARP1/2 have been postulated to have a role in protecting the ends and preventing the recruitment of KU, thereby promoting the alternative mechanisms and resection.

Alt-NHEJ is cell cycle independent; it can occur in G1, where limited resection is present and exposes the micro-homology or, alternatively, a helicase might expose the microhomology (as occurs in MMEJ). Polymerases can fill in the gap and the XPF/ERCC1 complex (which is an endonuclease component also involved in NER and SSA) has a role in removing the DNA flap (the displaced strand that gets created). Finally, ligase I and a complex of XRCC1 and ligase III appear to have a role in the ligation of the ends. The latter two factors are also involved in NER, BER and SSBR.

Microhomology Mediated End-Joining (MMEJ)

In some embodiments, MMEJ is down-regulated in order to promote HDR. MMEJ is a type of alt-NHEJ. MMEJ typically acts where there has been a small degree of resection (e.g., 5-25 nt) at the break. It may be considered a backup pathway for situations where NHEJ fails.

The initial phase of MMEJ involves recognition of the break. PARP1/2, which binds to double strand breaks, can promote MMEJ. Next, the cell performs resection over a short distance from the break site. CtIP performs some resection in G1, which can also promote MMEJ. Next, the single stranded microhomology domains anneal with each other and LIG-3 performs DNA end ligation.

Synthesis Dependent Microhomology Mediated End-Joining (SSMMEJ)

In some embodiments, SDMMEJ is down-regulated in order to promote HDR. One of the best-reported proteins involved in SDMMEJ is Polymerase Theta (Pol Theta). Accordingly, in some embodiments, Pol Theta is inhibited in order to promote HDR.

In some embodiments, the methods herein involve down-regulating alt-NHEJ, e.g., MMEJ and/or SDMMEJ, in order to promote HDR. For instance, the methods may involve modulating, e.g., inhibiting, a component (e.g., exactly one component, or one or more components, e.g., two or three components) of the MMEJ pathway, e.g., a component of Table VI.9 or Table VI.1(J). More specifically, the methods may involve modulating, e.g., inhibiting, exactly one of, or one or more of a PARP, PARP1, PARP2, CtIP, and LIG-3. In another embodiment, the methods may involve modulating, e.g., inhibiting, Pol Theta. Up-regulation of HDR, in some embodiments, is achieved using siRNA directed against one or more of the components listed in Table VI.9 or Table VI.1(J). In some embodiments, an alt-NHEJ pathway is down regulated using an HDR-enhancing gRNA that targets a component of an alt-NHEJ pathway, e.g., one or more of the components listed in Table VI.9 or Table VI.1(J) (e.g., a PARP, PARP1, PARP2, CtIP, and LIG-3). In some embodiments, the HDR-enhancing gRNA is used in combination with an eiCas9 molecule. In some embodiments, the HDR-enhancing gRNA is used in combination with an eaCas9 molecule. In some embodiments, the HDR-enhancing gRNA is administered to the cell as a HDR-enhancing gRNA:Cas9 molecule complex.

In embodiments, one or more of HDR, alt-HR, anti-HR, NHEJ, SSA, SSBR, MMR, NER, and BER are not substantially down-regulated, e.g., the only DNA damage repair pathway to be substantially down-regulated is an alt-NHEJ pathway such as MMEJ pathway and/or SDMMEJ.

TABLE VI.9 Factors that promote alt-NHEJ a PARP — PARP1 See Table VI.2 PARP2 See Table VI.2 CtIP See Table VI.2 LIG-3 MSLAFKIFFPQTLRALSRKELCLFRKHHWRDVRQFSQWSETDLLHGHPLFLRRKPVLSFQGSHLRSRATY LVFLPGLHVGLCSGPCEMAEQRFCVDYAKRGTAGCKKCKEKIVKGVCRIGKVVPNPFSESGGDMKEWYHI KCMFEKLERARATTKKIEDLTELEGWEELEDNEKEQITQHIADLSSKAAGTPKKKAVVQAKLTTTGQVTS PVKGASFVTSTNPRKFSGFSAKPNNSGEAPSSPTPKRSLSSSKCDPRHKDCLLREFRKLCAMVADNPSYN TKTQIIQDFLRKGSAGDGFHGDVYLTVKLLLPGVIKTVYNLNDKQIVKLFSRIFNCNPDDMARDLEQGDV SETIRVFFEQSKSFPPAAKSLLTIQEVDEFLLRLSKLTKEDEQQQALQDIASRCTANDLKCIIRLIKHDL KMNSGAKHVLDALDPNAYEAFKASRNLQDVVERVLHNAQEVEKEPGQRRALSVQASLMTPVQPMLAEACK SVEYAMKKCPNGMFSEIKYDGERVQVHKNGDHFSYFSRSLKPVLPHKVAHFKDYIPQAFPGGHSMILDSE VLLIDNKTGKPLPFGTLGVHKKAAFQDANVCLFVFDCIYFNDVSLMDRPLCERRKFLHDNMVEIPNRIMF SEMKRVTKALDLADMITRVIQEGLEGLVLKDVKGTYEPGKRHWLKVKKDYLNEGAMADTADLVVLGAFYG QGSKGGMMSIFLMGCYDPGSQKWCTVTKCAGGHDDATLARLQNELDMVKISKDPSKIPSWLKVNKIYYPD FIVPDPKKAAVWEITGAEFSKSEAHTADGISIRFPRCTRIRDDKDWKSATNLPQLKELYQLSKEKADFTV VAGDEGSSTTGGSSEENKGPSGSAVSRKAPSKPSASTKKAEGKLSNSNSKDGNMQTAKPSAMKVGEKLAT KSSPVKVGEKRKAADETLCQTKRRPASEQRGRTVPAGRR (LIG3 sequence 1) (SEQ ID NO: 304) MSLAFKIFFPQTLRALSRKELCLFRKHHWRDVRQFSQWSETDLLHGHPLFLRRKPVLSFQGSHLRSRATY LVFLPGLHVGLCSGPCEMAEQRFCVDYAKRGTAGCKKCKEKIVKGVCRIGKVVPNPFSESGGDMKEWYHI KCMFEKLERARATTKKIEDLTELEGWEELEDNEKEQITQHIADLSSKAAGTPKKKAVVQAKLTTTGQVTS PVKGASFVTSTNPRKFSGFSAKPNNSGEAPSSPTPKRSLSSSKCDPRHKDCLLREFRKLCAMVADNPSYN TKTQIIQDFLRKGSAGDGFHGDVYLTVKLLLPGVIKTVYNLNDKQIVKLFSRIFNCNPDDMARDLEQGDV SETIRVFFEQSKSFPPAAKSLLTIQEVDEFLLRLSKLTKEDEQQQALQDIASRCTANDLKCIIRLIKHDL KMNSGAKHVLDALDPNAYEAFKASRNLQDVVERVLHNAQEVEKEPGQRRALSVQASLMTPVQPMLAEACK SVEYAMKKCPNGMFSEIKYDGERVQVHKNGDHFSYFSRSLKPVLPHKVAHFKDYIPQAFPGGHSMILDSE VLLIDNKTGKPLPFGTLGVHKKAAFQDANVCLFVFDCIYFNDVSLMDRPLCERRKFLHDNMVEIPNRIMF SEMKRVTKALDLADMITRVIQEGLEGLVLKDVKGTYEPGKRHWLKVKKDYLNEGAMADTADLVVLGAFYG QGSKGGMMSIFLMGCYDPGSQKWCTVTKCAGGHDDATLARLQNELDMVKISKDPSKIPSWLKVNKIYYPD FIVPDPKKAAVWEITGAEFSKSEAHTADGISIRFPRCTRIRDDKDWKSATNLPQLKELYQLSKEKADFTV VAGDEGSSTTGGSSEENKGPSGSAVSRKAPSKPSASTKKAEGKLSNSNSKDGNMQTAKPSAMKVGEKLAT KSSPVKVGEKRKAADETLCQTKVLLDIFTGVRLYLPPSTPDFSRLRRYFVAFDGDLVQEFDMTSATHVLG SRDKNPAAQQVSPEWIWACIRKRRLVAPC (LIG3 sequence 2) (SEQ ID NO: 305) MVDVLLLFSLCLLFHISRPDLSHNRLSFIKASSMSHLQSLREVKLNNNELETIPNLGPVS ANITLLSLAGNRIVEILPEHLKEFQSLETLDLSSNNISELQTAFPALQLKYLYLNSNRVT SMEPGYFDNLANTLLVLKLNRNRISAIPPKMFKLPQLQHLELNRNKIKNVDGLTFQGLGA LKSLKMQRNGVTKLMDGAFWGLSNMEILQLDHNNLTEITKGWLYGLLMLQELHLSQNAIN RISPDAWEFCQKLSELDLTFNHLSRLDDSSFLGLSLLNTLHIGNNRVSYIADCAFRGLSS LKTLDLKNNEISWTIEDMNGAFSGLDKLRRLILQGNRIRSITKKAFTGLDALEHLDLSDN AIMSLQGNAFSQMKKLQQLHLNTSSLLCDCQLKWLPQWVAENNFQSFVNASCAHPQLLKG RSIFAVSPDGFVCDDFPKPQITVQPETQSAIKGSNLSFICSAASSSDSPMTFAWKKDNEL LHDAEMENYAHLRAQGGEVMEYTTILRLREVEFASEGKYQCVISNHFGSSYSVKAKLTVN MLPSFTKTPMDLTIRAGAMARLECAAVGHPAPQIAWQKDGGTDFPAARERRMHVMPEDDV FFIVDVKIEDIGVYSCTAQNSAGSISANATLTVLETPSFLRPLLDRTVTKGETAVLQCIA GGSPPPKLNWTKDDSPLVVTERHFFAAGNQLLIIVDSDVSDAGKYTCEMSNTLGTERGNV RLSVIPTPTCDSPQMTAPSLDDDGWATVGVVIIAVVCCVVGTSLVWVVIIYHTRRRNEDC SITNTDETNLPADIPSYLSSQGTLADRQDGYVSSESGSHHQFVTSSGAGFFLPQHDSSGT CHIDNSSEADVEAATDLFLCPFLGSTGPMYLKGNVYGSDPFETYHTGCSPDPRTVLMDHY EPSYIKKKECYPCSHPSEESCERSFSNISWPSHVRKLLNTSYSHNEGPGMKNLCLNKSSL DFSANPEPASVASSNSFMGTFGKALRRPHLDAYSSFGQPSDCQPRAFYLKAHSSPDLDSG SEEDGKERTDFQEENHICTFKQTLENYRTPNFQSYDLDT (LIG3 sequence 3) (SEQ ID NO: 306) MRE11 See Table VI.2 Rad50 See Table VI.2 Nbs1 See Table VI.2 CtIP See Table VI.2 XPF MESGQPARRIAMAPLLEYERQLVLELLDTDGLVVCARGLGADRLLYHFLQLHCHPACLVL VLNTQPAEEEYFINQLKIEGVEHLPRRVTNEITSNSRYEVYTQGGVIFATSRILVVDFLT DRIPSDLITGILVYRAHRIIESCQEAFILRLFRQKNKRGFIKAFTDNAVAFDTGFCHVER VMRNLFVRKLYLWPRFHVAVNSFLEQHKPEVVEIHVSMTPTMLAIQTAILDILNACLKEL KCHNPSLEVEDLSLENAIGKPFDKTIRHYLDPLWHQLGAKTKSLVQDLKILRTLLQYLSQ YDCVTFLNLLESLRATEKAFGQNSGWLFLDSSTSMFINARARVYHLPDAKMSKKEKISEK MEIKEGEETKKELVLESNPKWEALTEVLKEIEAENKESEALGGPGQVLICASDDRTCSQL RDYITLGAEAFLLRLYRKTFEKDSKAEEVWMKFRKEDSSKRIRKSHKRPKDPQNKERAST KERTLKKKKRKLTLTQMVGKPEELEEEGDVEEGYRREISSSPESCPEEIKHEEFDVNLSS DAAFGILKEPLTIIHPLLGCSDPYALTRVLHEVEPRYVVLYDAELTFVRQLEIYRASRPG KPLRVYFLIYGGSTEEQRYLTALRKEKEAFEKLIREKASMVVPEEREGRDETNLDLVRGT ASADVSTDTRKAGGQEQNGTQQSIVVDMREFRSELPSLIHRRGIDIEPVTLEVGDYILTP EMCVERKSISDLIGSLNNGRLYSQCISMSRYYKRPVLLIEFDPSKPFSLTSRGALFQEIS SNDISSKLTLLTLHFPRLRILWCPSPHATAELFEELKQSKPQPDAATALAITADSETLPE SEKYNPGPQDFLLKMPGVNAKNCRSLMHHVKNIAELAALSQDELTSILGNAANAKQLYDF IHTSFAEVVSKGKGKK (XPF CCDS 32390.1) (SEQ ID NO: 307) ERCC1 >sp|P07992|ERCC1_HUMAN DNA excision repair protein ERCC-1 OS = Homo sapiens GN = ERCC1 PE = 1 SV =  1 (SEQ ID NO: 308) MDPGKDKEGVPQPSGPPARKKFVIPLDEDEVPPGVAKPLFRSTQSLPTVDTSAQAAPQTY AEYAISQPLEGAGATCPTGSEPLAGETPNQALKPGAKSNSIIVSPRQRGNPVLKFVRNVP WEFGDVIPDYVLGQSTCALFLSLRYHNLHPDYIHGRLQSLGKNFALRVLLVQVDVKDPQQ ALKELAKMCILADCTLILAWSPEEAGRYLETYKAYEQKPADLLMEKLEQDFVSRVTECLT TVKSVNKTDSQTLLTTFGSLEQLIAASREDLALCPGLGPQKARRLFDVLHEPFLKVP >sp|P07992-2|ERCC1_HUMAN Isoform 2 of DNA excision repair protein ERCC-1 OS = Homo sapiens GN = ERCC1 (SEQ ID NO: 309) MDPGKDKEGVPQPSGPPARKKFVIPLDEDEVPPGVAKPLFRSTQSLPTVDTSAQAAPQTY AEYAISQPLEGAGATCPTGSEPLAGETPNQALKPGAKSNSIIVSPRQRGNPVLKFVRNVP WEFGDVIPDYVLGQSTCALFLSLRYHNLHPDYIHGRLQSLGKNFALRVLLVQVDVKDPQQ ALKELAKMCILADCTLILAWSPEEAGRYLETYKAYEQKPADLLMEKLEQDFVSRSLEQLI AASREDLALCPGLGPQKARRLFDVLHEPFLKVP >sp|P07992-3|ERCC1_HUMAN Isoform 3 of DNA excision repair protein ERCC-1 OS = Homo sapiens GN = ERCC1 (SEQ ID NO: 310) MDPGKDKEGVPQPSGPPARKKFVIPLDEDEVPPGVAKPLFRSTQSLPTVDTSAQAAPQTY AEYAISQPLEGAGATCPTGSEPLAGETPNQALKPGAKSNSIIVSPRQRGNPVLKFVRNVP WEFGDVIPDYVLGQSTCALFLSLRYHNLHPDYIHGRLQSLGKNFALRVLLVQVDVKDPQQ ALKELAKMCILADCTLILAWSPEEAGRYLETYKAYEQKPADLLMEKLEQDFVSRVTECLT TVKSVNKTDSQTLLTTFGSLEQLIAASREDLALCPGLGPQKVRALGKNPRSWGKERAPNK HNLRPQSFKVKKEPKTRHSGFRL >sp|P07992-4|ERCC1_HUMAN Isoform 4 of DNA excision repair protein ERCC-1 OS = Homo sapiens GN = ERCC1 (SEQ ID NO: 311) MDPGKDKEGVPQPSGPPARKKFVIPLDEDEVPPGVRGNPVLKFVRNVPWEFGDVIPDYVL GQSTCALFLSLRYHNLHPDYIHGRLQSLGKNFALRVLLVQVDVKDPQQALKELAKMCILA DCTLILAWSPEEAGRYLETYKAYEQKPADLLMEKLEQDFVSRVTECLTTVKSVNKTDSQT LLTTFGSLEQLIAASREDLALCPGLGPQKARRLFDVLHEPFLKVP Ligase-1 (LIG1) MQRSIMSFFHPKKEGKAKKPEKEASNSSRETEPPPKAALKEWNGVVSESDSPVKRPGRKAARVLGSEGEE EDEALSPAKGQKPALDCSQVSPPRPATSPENNASLSDTSPMDSSPSGIPKRRTARKQLPKRTIQEVLEEQ SEDEDREAKRKKEEEEEETPKESLTEAEVATEKEGEDGDQPTTPPKPLKTSKAETPTESVSEPEVATKQE LQEEEEQTKPPRRAPKTLSSFFTPRKPAVKKEVKEEEPGAPGKEGAAEGPLDPSGYNPAKNNYHPVEDAC WKPGQKVPYLAVARTFEKIEEVSARLRMVETLSNLLRSVVALSPPDLLPVLYLSLNHLGPPQQGLELGVG DGVLLKAVAQATGRQLESVRAEAAEKGDVGLVAENSRSTQRLMLPPPPLTASGVFSKFRDIARLTGSAST AKKIDIIKGLFVACRHSEARFIARSLSGRLRLGLAEQSVLAALSQAVSLTPPGQEFPPAMVDAGKGKTAE ARKTWLEEQGMILKQTFCEVPDLDRIIPVLLEHGLERLPEHCKLSPGIPLKPMLAHPTRGISEVLKRFEE AAFTCEYKYDGQRAQIHALEGGEVKIFSRNQEDNTGKYPDIISRIPKIKLPSVTSFILDTEAVAWDREKK QIQPFQVLTTRKRKEVDASEIQVQVCLYAFDLIYLNGESLVREPLSRRRQLLRENFVETEGEFVFATSLD TKDIEQIAEFLEQSVKDSCEGLMVKTLDVDATYEIAKRSHNWLKLKKDYLDGVGDTLDLVVIGAYLGRGK RAGRYGGFLLASYDEDSEELQAICKLGTGFSDEELEEHHQSLKALVLPSPRPYVRIDGAVIPDHWLDPSA VWEVKCADLSLSPIYPAARGLVDSDKGISLRFPRFIRVREDKQPEQATTSAQVACLYRKQSQIQNQQGED SGSDPEDTY (LIG1 sequence 1) (SEQ ID NO: 312) MQRSIMAALKEWNGVVSESDSPVKRPGRKAARVLGSEGEEEDEALSPAKGQKPALDCSQVSPPRPATSPE NNASLSDTSPMDSSPSGIPKRRTARKQLPKRTIQEVLEEQSEDEDREAKRKKEEEEETPKESLTEAEVAT EKEGEDGDQPTTPPKPLKTSKAETPTESVSEPEVATKQELQEEEEQTKPPRRAPKTLSSFFTPRKPAVKK EVKEEEPGAPGKEGAAEGPLDPSGYNPAKNNYHPVEDACWKPGQKVPYLAVARTFEKIEEVSARLRMVET LSNLLRSVVALSPPDLLPVLYLSLNHLGPPQQGLELGVGDGVLLKAVAQATGRQLESVRAEAAEKGDVGL VAENSRSTQRLMLPPPPLTASGVFSKFRDIARLTGSASTAKKIDIIKGLFVACRHSEARFIARSLSGRLR LGLAEQSVLAALSQAVSLTPPGQEFPPAMVDAGKGKTAEARKTWLEEQGMILKQTFCEVPDLDRIIPVLL EHGLERLPEHCKLSPGIPLKPMLAHPTRGISEVLKRFEEAAFTCEYKYDGQRAQIHALEGGEVKIFSRNQ EDNTGKYPDIISRIPKIKLPSVTSFILDTEAVAWDREKKQIQPFQVLTTRKRKEVDASEIQVQVCLYAFD LIYLNGESLVREPLSRRRQLLRENFVETEGEFVFATSLDTKDIEQIAEFLEQSVKDSCEGLMVKTLDVDA TYEIAKRSHNWLKLKKDYLDGVGDILDLVVIGAYLGRGKRAGRYGGFLLASYDEDSEELQAICKLGTGFS DEELEEHHQSLKALVLPSPRPYVRIDGAVIPDHWLDPSAVWEVKCADLSLSPIYPAARGLVDSDKGISLR FPRFIRVREDKQPEQATTSAQVACLYRKQSQIQNQQGEDSGSDPEDTY (LIG1 sequence 2) (SEQ ID NO: 313) MQRSIMSFFHPKKEGKAKKPEKEASNSSRETEPPPKAALKEWNGVVSESDSPVKRPGRKAARVLGSEGEE EDEALSPAKGQKPALDCSQVSPPRPATSPENNASLSDTSPMDSSPSGIPKRRTEAETPTESVSEPEVATK QELQEEEEQTKPPRRAPKTLSSFFTPRKPAVKKEVKEEEPGAPGKEGAAEGPLDPSGYNPAKNNYHPVED ACWKPGQKVPYLAVARTFEKIEEVSARLRMVETLSNLLRSVVALSPPDLLPVLYLSLNHLGPPQQGLELG VGDGVLLKAVAQATGRQLESVRAEAAEKGDVGLVAENSRSTQRLMLPPPPLTASGVFSKFRDIARLTGSA STAKKIDIIKGLFVACRHSEARFIARSLSGRLRLGLAEQSVLAALSQAVSLTPPGQEFPPAMVDAGKGKT AEARKTWLEEQGMILKQTFCEVPDLDRIIPVLLEHGLERLPEHCKLSPGIPLKPMLAHPTRGISEVLKRF EEAAFTCEYKYDGQRAQIHALEGGEVKIFSRNQEDNTGKYPDIISRIPKIKLPSVTSFILDTEAVAWDRE KKQIQPFQVLTTRKRKEVDASEIQVQVCLYAFDLIYLNGESLVREPLSRRRQLLRENFVETEGEFVFATS LDTKDIEQIAEFLEQSVKDSCEGLMVKTLDVDATYEIAKRSHNWLKLKKDYLDGVGDTLDLVVIGAYLGR GKRAGRYGGFLLASYDEDSEELQAICKLGTGFSDEELEEHHQSLKALVLPSPRPYVRIDGAVIPDHWLDP SAVWEVKCADLSLSPIYPAARGLVDSDKGISLRFPRFIRVREDKQPEQATTSAQVACLYRKQSQIQNQQG EDSGSDPEDTY (LIG1 sequence 3) (SEQ ID NO: 314) Pol Theta >sp|O75417|DPOLQ_HUMAN DNA polymerase theta OS = Homo sapiens GN = POLQ PE = 1 SV = 2 (SEQ ID NO: 315) MNLLRRSGKRRRSESGSDSFSGSGGDSSASPQFLSGSVLSPPPGLGRCLKAAAAGECKPT VPDYERDKLLLANWGLPKAVLEKYHSFGVKKMFEWQAECLLLGQVLEGKNLVYSAPTSAG KTLVAELLILKRVLEMRKKALFILPFVSVAKEKKYYLQSLFQEVGIKVDGYMGSTSPSRH FSSLDIAVCTIERANGLINRLIEENKMDLLGMVVVDELHMLGDSHRGYLLELLLTKICYI TRKSASCQADLASSLSNAVQIVGMSATLPNLELVASWLNAELYHTDFRPVPLLESVKVGN SIYDSSMKLVREFEPMLQVKGDEDHVVSLCYETICDNHSVLLFCPSKKWCEKLADIIARE FYNLHHQAEGLVKPSECPPVILEQKELLEVMDQLRRLPSGLDSVLQKTVPWGVAFHHAGL TFEERDIIEGAFRQGLIRVLAATSTLSSGVNLPARRVIIRTPIFGGRPLDILTYKQMVGR AGRKGVDTVGESILICKNSEKSKGIALLQGSLKPVRSCLQRREGEEVTGSMIRAILEIIV GGVASTSQDMHTYAACTFLAASMKEGKQGIQRNQESVQLGAIEACVMWLLENEFIQSTEA SDGTEGKVYHPTHLGSAILSSSLSPADTLDIFADLQRAMKGFVLENDLHILYLVIPMFED WITIDWYRFFCLWEKLPTSMKRVAELVGVEEGFLARCVKGKVVARTERQHRQMAIHKRFF TSLVLLDLISEVPLREINQKYGCNRGQIQSLQQSAAVYAGMITVFSNRLGWHNMELLLSQ FQKRLIFGIQRELCDLVRVSLLNAQRARVLYASGFHTVADLARANIVEVEVILKNAVPFK SARKAVDEEEEAVEERRNMRTIWVTGRKGLTEREAAALIVEEARMILQQDLVEMGVQWNP CALLHSSICSLTHSESEVKEHTFISQTKSSYKKLISKNKSNTIFSDSYIKHSPNIVQDLN KSREHTSSFNCNFQNGNQEHQICSIFRARKRASLDINKEKPGASQNEGKISDKKVVQTFS QKIKKAPLNFNSEKMSRSFRSWKRRKHLKRSRDSSPLKDSGACRIHLQGQILSNPSLCED PFILDEKKTEFRNSGPFAKNVSLSGKEKDNKTSFPLQIKQNCSWNITLINDNFVEHIVIG SQSKNVICQATSVVSEKGRGVAVEAEKINEVLIQNGSKNQNVYMKHHDIHPINQYLRKQS HEQTSTITKQKNIIERQMPCEAVSSYINRDSNVTINCERIKLNTEENKPSHFQALGDDIS RIVIPSEVLPSAGAFSKSEGQHENFLNISRLQEKTGTYTINKTKNNHVSDLGLVLCDFED SFYLDTQSEKIIQQMATENAKLGAKDINLAAGIMQKSLVQQNSMNSFQKECHIPFPAEQH PLGATKIDHLDLKTVGIMKQSSDSHGVDILTPESPIFHSPILLEENGLFLKKNEVSVIDS QLNSFLQGYQTQETVKPVILLIPQKRIPTGVEGECLPVPETSLNMSDSLLFDSFSDDYLV KEQLPDMQMKEPLPSEVISNHFSDSLCLQEDLIKKSNVNENQDTHQQLICSNDESIIFSE MDSVQMVEALDNVDIFPVQEKNHTVVSPRALELSDPVLDEHHQGDQDGGDQDERAEKSKL TGIRQNHSFIWSGASFDLSPGLQRILDKVSSPLENEKLKSMTINFSSLNRKNTELNEEQE VISNLETKQVQGISFSSNNEVKSKIEMLENNANHDETSSLLPRKESNIVDDNGLIPPIPI PTSASKLIFPGILETPVNPWKINNVLQPGESYLFGSPSDIKNHDLSPGSRNGFKDNSPIS DISFSLQLSQDGLQLTPASSSSESLSIIDVASDQNLFQTFIKEWRCKKRFSISLACEKIR SLISSKTATIGSRFKQASSPQEIPIRDDGFPIKGCDDILVVGLAVCWGGRDAYYFSLQKE QKHSEISASLVPPSLDPSLILKDRMWYLQSCLRKESDKECSVVIYDFIQSYKILLLSCGI SLEQSYEDPKVACWLLDPDSQEPTLHSIVISFLPHELPLLEGMETSQGIQSLGLNAGSEH SGRYRASVESILIFNSMNQLNSLLQKENLQDVFRKVEMPSQYCLALLELNGIGFSTAECE SQKHIMQAKLDAIETQAYQLAGHSFSFISSDDIAEVLFLELKLPPNREMKNQGSKKILGS TRRGIDNGRKLRLGRQFSTSKDVLNKLKALHPLPGLILEWRRITNAITKVVFPLQREKCL NPFLGMERIYPVSQSHTATGRITFTEPNIQNVPRDFEIKMPTLVGESPPSQAVGKGLLPM GRGKYKKGFSVNPRCQAQMEERAADRGMPFSISMRHAFVPFPGGSILAADYSQLELRILA HLSHDRRLIQVLNIGADVFRSIAAEWKMIEPESVGDDLRQQAKQICYGIIYGMGAKSLGE QMGIKENDAACYIDSFKSRYTGINQFMTETVKNCKRDGFVQTILGRRRYLPGIKDNNPYR KAHAERQAINTIVQGSAADIVKIATVNIQKQLETFHSTFKSHGHREGMLQSDQTGLSRKR KLQGMFCPIRGGFFILQLHDELLYEVAEEDVVQVAQIVKNEMESAVKLSVKLKVKVKIGA SWGELKDFDV >sp|O75417-2|DPOLQ_HUMAN Isoform 2 of DNA polymerase theta OS = Homo sapiens GN = POLQ (SEQ ID NO: 316) MNSFLSFPISLCSARKAVDEEEEAVEERRNMRTIWVTGRKGLTEREAAALIVEEARMILQ QDLVEMGVQWNPCALLHSSICSLTHSESEVKEHTFISQTKSSYKKLISKNKSNTIFSDSY IKHSPNIVQDLNKSREHTSSFNCNFQNGNQEHQICSIFRARKRASLDINKEKPGASQNEG KTSDKKVVQTFSQKIKKAPLNFNSEKMSRSFRSWKRRKHLKRSRDSSPLKDSGACRIHLQ GQILSNPSLCEDPFILDEKKTEFRNSGPFAKNVSLSGKEKDNKTSFPLQIKQNCSWNITL INDNFVEHIVIGSQSKNVICQATSVVSEKGRGVAVEAEKINEVLIQNGSKNQNVYMKHHD IHPINQYLRKQSHEQTSTITKQKNIIERQMPCEAVSSYINRDSNVTINCERIKLNTEENK PSHFQALGDDISRIVIPSEVLPSAGAFSKSEGQHENFLNISRLQEKTGTYTINKTKNNHV SDLGLVLCDFEDSFYLDTQSEKIIQQMATENAKLGAKDINLAAGIMQKSLVQQNSMNSFQ KECHIPFPAEQHPLGATKIDHLDLKTVGIMKQSSDSHGVDILTPESPIFHSPILLEENGL FLKKNEVSVTDSQLNSFLQGYQTQETVKPVILLIPQKRTPTGVEGECLPVPETSLNMSDS LLFDSFSDDYLVKEQLPDMQMKEPLPSEVTSNHFSDSLCLQEDLIKKSNVNENQDTHQQL TCSNDESIIFSEMDSVQMVEALDNVDIFPVQEKNHTVVSPRALELSDPVLDEHHQGDQDG GDQDERAEKSKLTGTRQNHSFIWSGASFDLSPGLQRILDKVSSPLENEKLKSMTINFSSL NRKNTELNEEQEVISNLETKQVQGISFSSNNEVKSKIEMLENNANHDETSSLLPRKESNI VDDNGLIPPTPIPTSASKLTFPGILETPVNPWKTNNVLQPGESYLFGSPSDIKNHDLSPG SRNGFKDNSPISDTSFSLQLSQDGLQLTPASSSSESLSIIDVASDQNLFQTFIKEWRCKK RFSISLACEKIRSLTSSKTATIGSRFKQASSPQEIPIRDDGFPIKGCDDTLVVGLAVCWG GRDAYYFSLQKEQKHSEISASLVPPSLDPSLTLKDRMWYLQSCLRKESDKECSVVIYDFI QSYKILLLSCGISLEQSYEDPKVACWLLDPDSQEPTLHSIVTSFLPHELPLLEGMETSQG IQSLGLNAGSEHSGRYRASVESILIFNSMNQLNSLLQKENLQDVFRKVEMPSQYCLALLE LNGIGFSTAECESQKHIMQAKLDAIETQAYQLAGHSFSFTSSDDIAEVLFLELKLPPNRE MKNQGSKKTLGSTRRGIDNGRKLRLGRQFSTSKDVLNKLKALHPLPGLILEWRRITNAIT KVVFPLQREKCLNPFLGMERTYPVSQSHTATGRITFTEPNIQNVPRDFEIKMPTLVGESP PSQAVGKGLLPMGRGKYKKGFSVNPRCQAQMEERAADRGMPFSISMRHAFVPFPGGSILA ADYSQLELRILAHLSHDRRLIQVLNTGADVFRSIAAEWKMIEPESVGDDLRQQAKQICYG ITYGMGAKSLGEQMGIKENDAACYIDSFKSRYTGINQFMTETVKNCKRDGFVQTILGRRR YLPGIKDNNPYRKAHAERQAINTIVQGSAADIVKIATVNIQKQLETFHSTFKSHGHREGM LQSDQTGLSRKRKLQGMFCPIRGGFFILQLHDELLYEVAEEDVVQVAQIVKNEMESAVKL SVKLKVKVKIGASWGELKDFDV MRN complex — XRCC1 >sp|P18887|XRCC1_HUMAN DNA repair protein XRCC1 OS = Homo sapiens GN = XRCC1 PE = 1 SV = 2 (SEQ ID NO: 317) MPEIRLRHVVSCSSQDSTHCAENLLKADTYRKWRAAKAGEKTISVVLQLEKEEQIHSVDI GNDGSAFVEVLVGSSAGGAGEQDYEVLLVTSSFMSPSESRSGSNPNRVRMFGPDKLVRAA AEKRWDRVKIVCSQPYSKDSPFGLSFVRFHSPPDKDEAEAPSQKVTVTKLGQFRVKEEDE SANSLRPGALFFSRINKTSPVTASDPAGPSYAAATLQASSAASSASPVSRAIGSTSKPQE SPKGKRKLDLNQEEKKTPSKPPAQLSPSVPKRPKLPAPTRTPATAPVPARAQGAVTGKPR GEGTEPRRPRAGPEELGKILQGVVVVLSGFQNPFRSELRDKALELGAKYRPDWTRDSTHL ICAFANTPKYSQVLGLGGRIVRKEWVLDCHRMRRRLPSRRYLMAGPGSSSEEDEASHSGG SGDEAPKLPQKQPQTKTKPTQAAGPSSPQKPPTPEETKAASPVLQEDIDIEGVQSEGQDN GAEDSGDTEDELRRVAEQKEHRLPPGQEENGEDPYAGSTDENTDSEEHQEPPDLPVPELP DFFQGKHFFLYGEFPGDERRKLIRYVTAFNGELEDNMSDRVQFVITAQEWDPSFEEALMD NPSLAFVRPRWIYSCNEKQKLLPHQLYGVVPQA

Inhibitors of some of the proteins of Table VI.9 above are listed elsewhere in this specification. In some embodiments alt-NHEJ is down-regulated using an agent of Table VI.10, or any combination thereof.

TABLE VI.10 Down-regulators of the alt-NHEJ pathway. L67 (inhibits LIG1 and LIG3; reviewed in Tomkinson et al. (2013) TRANSL. CANCER RES. 2(3): 1213.) L82 (inhibits LIG1; reviewed in Tomkinson et al.) L189 (inhibits LIG1, LIG3, and LIG4; reviewed in Tomkinson et al.) SCR7 (inhibits LIG3 and LIG4; reviewed in Tomkinson et al.)

VI.5 Single Strand Annealing (SSA)

Single strand annealing (SSA) is a that repairs double-stranded breaks. SSA is believed to be a sub-branch of HDR. As with HDR, a cell typically uses SSA when there has been significant resection at the break. Thus, SSA is characterized by having longer length of resection (longer than Alt-NHEJ) and a longer stretch of homology at the DSB site (>30 bp). SSA competes with HR in S phase.

As in other HDR pathways, resection leads to the formation of single stranded DNA regions. These regions are bound and stabilized by RPA, a heterotrimer comprising RPA1, RPA2, and RPA3. Whereas in the other HDR pathways, RAD51 binds the single stranded region, in the SSA pathway, RAD52 is involved. RAD52 promotes annealing of the two single stranded DNA segments at repetitive regions. Next, XPF/ERCC1 removes DNA flaps to make the DNA more suitable for ligation.

In some embodiments, other HDR pathways are promoted by down-regulating the SSA pathway. For example, in some embodiments, a Cas9 molecule and a gRNA can induce a DSB in a desired location during G2 or another phase of the cell cycle. This DSB can be formed using, e.g., one Cas9 molecule with the ability to produce DSBs, or two nickases. The DSB may undergo some resection, and/or may be created by a pair of staggered nicks that leaves some single stranded DNA at the break. In S/G2, an SSA down-regulator can increase the likelihood that a resected DSB is repaired by HDR. A template nucleic acid can be added to the cell, so that the HDR machinery repairs the DSB using the template nucleic acid.

Accordingly, in some embodiments, the methods herein involve down-regulating SSA in order to promote HDR pathways, such as HR and/or alt-HR. For instance, the methods may involve modulating, e.g., inhibiting, a component (e.g., exactly one component, or one or more components, e.g., two or three components) of the SSA pathway, e.g., a component of Table VI.1(E) or Table VI.11. More specifically, the methods may involve modulating, e.g., inhibiting, exactly one of, or one or more of RPA, RPA1, RPA2, RPA3, RAD52, XPF/ERCC1, and a ligase. Up-regulation of HDR, in some embodiments, is achieved using siRNA directed against one or more of the components listed in Table VI.1(E) or Table VI.11. In some embodiments, a SSA pathway is down regulated using an HDR-enhancing gRNA that targets a component of a SSA pathway, e.g., one or more of the components listed in Table VI.1(E) or Table VI.11 (e.g., RPA, RPA1, RPA2, RPA3, RAD52, XPF/ERCC1, and a ligase). In some embodiments, the HDR-enhancing gRNA is used in combination with an eiCas9 molecule. In some embodiments, the HDR-enhancing gRNA is used in combination with an eaCas9 molecule. In some embodiments, the HDR-enhancing gRNA is administered to the cell as a HDR-enhancing gRNA:Cas9 molecule complex.

In some embodiments, one or more of HR, alt-HR, anti-HR, NHEJ, MMEJ, SSBR, MMR, NER, and BER are not substantially down-regulated, e.g., the only DNA damage repair pathway to be substantially down-regulated is the SSA pathway.

TABLE VI.11 Factors that promote SSA RPA — RPA1 >sp|095602|RPA1_HUMAN DNA-directed RNA polymerase I subunit RPA1 OS = Homo sapiens GN = POLR1A PE = 1 SV = 2 (SEQ ID NO: 318) MLISKNMPWRRLQGISFGMYSAEELKKLSVKSITNPRYLDSLGNPSANGLYDLALGPADS KEVCSTCVQDFSNCSGHLGHIELPLTVYNPLLFDKLYLLLRGSCLNCHMLTCPRAVIHLL LCQLRVLEVGALQAVYELERILNRFLEENPDPSASEIREELEQYTTEIVQNNLLGSQGAH VKNVCESKSKLIALFWKAHMNAKRCPHCKTGRSVVRKEHNSKLTITFPAMVHRTAGQKDS EPLGIEEAQIGKRGYLTPTSAREHLSALWKNEGFFLNYLFSGMDDDGMESRFNPSVFFLD FLVVPPSRYRPVSRLGDQMFTNGQTVNLQAVMKDVVLIRKLLALMAQEQKLPEEVATPTT DEEKDSLIAIDRSFLSTLPGQSLIDKLYNIWIRLQSHVNIVFDSEMDKLMMDKYPGIRQI LEKKEGLFRKHMMGKRVDYAARSVICPDMYINTNEIGIPMVFATKLTYPQPVTPWNVQEL RQAVINGPNVHPGASMVINEDGSRTALSAVDMTQREAVAKQLLTPATGAPKPQGTKIVCR HVKNGDILLLNRQPTLHRPSIQAHRARILPEEKVLRLHYANCKAYNADFDGDEMNAHFPQ SELGRAEAYVLACTDQQYLVPKDGQPLAGLIQDHMVSGASMTTRGCFFTREHYMELVYRG LTDKVGRVKLLSPSILKPFPLWTGKQVVSTLLINIIPEDHIPLNLSGKAKITGKAWVKET PRSVPGFNPDSMCESQVIIREGELLCGVLDKAHYGSSAYGLVHCCYEIYGGETSGKVLTC LARLFTAYLQLYRGFTLGVEDILVKPKADVKRQRIIEESTHCGPQAVRAALNLPEAASYD EVRGKWQDAHLGKDQRDFNMIDLKFKEEVNHYSNEINKACMPFGLHRQFPENSLQMMVQS GAKGSTVNTMQISCLLGQIELEGRRPPLMASGKSLPCFEPYEFTPRAGGFVTGRFLTGIK PPEFFFHCMAGREGLVDTAVKTSRSGYLQRCIIKHLEGLVVQYDLTVRDSDGSVVQFLYG EDGLDIPKTQFLQPKQFPFLASNYEVIMKSQHLHEVLSRADPKKALHHFRAIKKWQSKHP NTLLRRGAFLSYSQKIQEAVKALKLESENRNGRSPGTQEMLRMWYELDEESRRKYQKKAA ACPDPSLSVWRPDIYFASVSETFETKVDDYSQEWAAQTEKSYEKSELSLDRLRTLLQLKW QRSLCEPGEAVGLLAAQSIGEPSTQMTLNTFHFAGRGEMNVTLGIPRLREILMVASANIK TPMMSVPVLNTKKALKRVKSLKKQLTRVCLGEVLQKIDVQESFCMEEKQNKFQVYQLRFQ FLPHAYYQQEKCLRPEDILRFMETRFFKLLMESIKKKNNKASAFRNVNTRRATQRDLDNA GELGRSRGEQEGDEEEEGHIVDAEAEEGDADASDAKRKEKQEEEVDYESEEEEEREGEEN DDEDMQEERNPHREGARKTQEQDEEVGLGTEEDPSLPALLTQPRKPTHSQEPQGPEAMER RVQAVREIHPFIDDYQYDTEESLWCQVTVKLPLMKINFDMSSLVVSLAHGAVIYATKGIT RCLLNETTNNKNEKELVLNTEGINLPELFKYAEVLDLRRLYSNDIHAIANTYGIEAALRV IEKEIKDVFAVYGIAVDPRHLSLVADYMCFEGVYKPLNRFGIRSNSSPLQQMTFETSFQF LKQATMLGSHDELRSPSACLVVGKVVRGGTGLFELKQPLR RPA2 >sp|P15927|RFA2_HUMAN Replication protein A 32 kDa subunit OS = Homo sapiens GN = RPA2 PE = 1 SV = 1 (SEQ ID NO: 319) MWNSGFESYGSSSYGGAGGYTQSPGGFGSPAPSQAEKKSRARAQHIVPCTISQLLSATLV DEVFRIGNVEISQVTIVGIIRHAEKAPTNIVYKIDDMTAAPMDVRQWVDTDDTSSENTVV PPETYVKVAGHLRSFQNKKSLVAFKIMPLEDMNEFTTHILEVINAHMVLSKANSQPSAGR APISNPGMSEAGNFGGNSFMPANGLTVAQNQVLNLIKACPRPEGLNFQDLKNQLKHMSVS SIKQAVDFLSNEGHIYSTVDDDHFKSTDAE >sp|P15927-2|RFA2_HUMAN Isoform 2 of Replication protein A 32 kDa subunit OS = Homo sapiens GN = RPA2 (SEQ ID NO: 320) MGRGDRNKRSIRGFESYGSSSYGGAGGYTQSPGGFGSPAPSQAEKKSRARAQHIVPCTIS QLLSATLVDEVFRIGNVEISQVTIVGIIRHAEKAPTNIVYKIDDMTAAPMDVRQWVDTDD TSSENTVVPPETYVKVAGHLRSFQNKKSLVAFKIMPLEDMNEFTTHILEVINAHMVLSKA NSQPSAGRAPISNPGMSEAGNFGGNSFMPANGLTVAQNQVLNLIKACPRPEGLNFQDLKN QLKHMSVSSIKQAVDFLSNEGHIYSTVDDDHFKSTDAE >sp|P15927-3|RFA2_HUMAN Isoform 3 of Replication protein A 32 kDa subunit OS = Homo sapiens GN = RPA2 (SEQ ID NO: 321) MWNSNDGGAGWRRKRIAGGFSKRASLGSERRVVAGEEGRERSWGVWGSPAGRRRGRLGRL GQCLKGRSLREPAGFSEAWDVAQALILLFKTGGFESYGSSSYGGAGGYTQSPGGFGSPAP SQAEKKSRARAQHIVPCTISQLLSATLVDEVFRIGNVEISQVTIVGIIRHAEKAPTNIVY KIDDMTAAPMDVRQWVDTDDTSSENTVVPPETYVKVAGHLRSFQNKKSLVAFKIMPLEDM NEFTTHILEVINAHMVLSKANSQPSAGRAPISNPGMSEAGNFGGNSFMPANGLTVAQNQV LNLIKACPRPEGLNFQDLKNQLKHMSVSSIKQAVDFLSNEGHIYSTVDDDHFKSTDAE RPA3 See Table VI.2 RAD52 >sp|P43351|RAD52_HUMAN DNA repair protein RAD52 homolog OS = Homo sapiens GN = RAD52 PE = 1 SV = 1 (SEQ ID NO: 322) MSGTEEAILGGRDSHPAAGGGSVLCFGQCQYTAEEYQAIQKALRQRLGPEYISSRMAGGG QKVCYIEGHRVINLANEMFGYNGWAHSITQQNVDFVDLNNGKFYVGVCAFVRVQLKDGSY HEDVGYGVSEGLKSKALSLEKARKEAVTDGLKRALRSFGNALGNCILDKDYLRSLNKLPR QLPLEVDLTKAKRQDLEPSVEEARYNSCRPNMALGHPQLQQVTSPSRPSHAVIPADQDCS SRSLSSSAVESEATHQRKLRQKQLQQQFRERMEKQQVRVSTPSAEKSEAAPPAPPVTHST PVTVSEPLLEKDFLAGVTQELIKTLEDNSEKWAVTPDAGDGVVKPSSRADPAQTSDTLAL NNQMVTQNRTPHSVCHQKPQAKSGSWDLQTYSADQRTTGNWESHRKSQDMKKRKYDPS >sp|P43351-2|RAD52_HUMAN Isoform beta of DNA repair protein RAD52 homolog OS = Homo sapiens GN = RAD52 (SEQ ID NO: 323) MSGTEEAILGGRDSHPAAGGGSVLCFGQCQYTAEEYQAIQKALRQRLGPEYISSRMAGGG QKVCYIEGHRVINLANEMFGYNGWAHSITQQNVDFVDLNNGKFYVGVCAFVRVQLKDGSY HEDVGYGVSEGLKSKALSLEKARKEAVTDGLKRALRLPLLGVSGRILYSLFSVHSVMCAG GLPTPTASAQTAPSSPCSSAVLRYAQEFWECTWKLYSGQRLPEITK >sp|P43351-3|RAD52_HUMAN Isoform gamma of DNA repair protein RAD52 homolog OS = Homo sapiens GN = RAD52 (SEQ ID NO: 324) MSGTEEAILGGRDSHPAAGGGSVLCFGQCQYTAEEYQAIQKALRQRLGPEYISSRMAGGG QKVCYIEGHRVINLANEMFGYNGWAHSITQQNVDFVDLNNGKFYVGVCAFVRVQLKVRGW SRPAARKDQWVVGEGWFIS >sp|P43351-4|RAD52_HUMAN Isoform delta of DNA repair protein RAD52 homolog OS = Homo sapiens GN = RAD52 (SEQ ID NO: 325) MSGTEEAILGGRDSHPAAGGGSVLCFGQCQYTAEEYQAIQKALRQRLGPEYISSRMAGGG QKVCYIEGHRVINLANEMFGYNGWAHSITQQNVGEYALQQWGLLHCPAPAESLLWVRR XPF See Table VI.9 ERCC1 See Table VI.9 a ligase —

More specifically, in some embodiments, the SSA down-regulator is an siRNA targeting Rad51, an siRNA targeting ERCC1, or an agent of Table VI.12 or any combination thereof. Inhibitors of some of the proteins of Table VI.11 above are also listed elsewhere in this specification.

TABLE VI.12 Down-regulators of SSA. 1. Rad52 Compounds Rad52 inhibitor deposited as PubChemAID: 651668 siRNA RAD52 Silencer, sold by Life Technologies; Accell Human RAD52 siRNA, sold by Dharmacon; Commercially available from Dharmacon or Ambion antibodies RAD52 antibody (C-term), sold by Abgent; RAD52 antibody, sold by Novus Biologicals 2. ERCC1 Compound Reference NSC 130813 Jordheim et al (2013) MOL. PHARMACOL 84(1): 12-24. siRNAs ERCC1 excision repair cross-complementation group 1 siRNA, sold by Dharmacon; ERCC1 Silencer, sold by Life Technologies; Commercially available from Dharmacon or Ambion antibodies ERCC1 Antibody #3885, sold by Cell Signaling Technology; ERCC1 Antibody (8F1), sold by Novus Biologicals 3. XPF Compounds Reference NSC 130813 Jordheim et al (2013) MOL. PHARMACOL. 84(1): 12-24. siRNAs Commercially available from Dharmacon or Ambion

VI.6 Single Strand Break Repair (SSBR)

Single-strand breaks (SSBs) in the genome are repaired by the SSBR pathway, which is a distinct mechanism from the DSB repair mechanisms discussed above (see FIG. 16 ). “SSBR”, as used herein, refers to a DNA repair process that has four major stages: SSB detection, DNA end processing, DNA gap filling, and DNA ligation (see, e.g., Caldecott (2008) NAT. REV. GENET. 9: 619-31). A brief summary of SSBR is provided below.

In the first stage, when a SSB forms, PARP1 and/or PARP2 recognize the break and recruit the repair machinery. The binding and activity of PARP1 at DNA breaks is transient and appears to accelerate SSBR by promoting the focal accumulation or stability of SSBR protein complexes at the lesion. Arguably the most important of these SSBR proteins is XRCC1, which functions as a molecular scaffold that interacts with, stabilizes, and stimulates multiple enzymatic components of the SSBRr process including the protein responsible for processing the DNA 3′ and 5′ ends. For instance, XRCC1 interacts with several proteins (DNA polymerase beta, PNK, and three nucleases, APE1, APTX, and APLF) that promote end processing. APE1 has endonuclease activity. APLF has endonuclease and 3′ to 5′ exonuclease activities. APTX has endonuclease and 3′ to 5′ exonuclease activity. This end processing is an important stage of SSBR since the 3′- and/or 5′-termini of most, if not all, SSBs are “damaged”. End processing generally involves restoring a damaged 3′ end to a hydroxylated state and/or restoring a damaged 5′ end to a phosphate moiety, thereby producing ligation-competent ends. Enzymes that process damaged 3′ termini include PNKP, APE1, and TDP1. Enzymes that process damaged 5′ termini include PNKP, DNA polymerase beta (Pol beta), and APTX. LIG3 (DNA ligase III) can also participate in end processing. Once the ends are cleaned, gap filling can occur.

At the DNA gap filling stage, the proteins typically present are PARP1, DNA polymerase beta, XRCC1, FEN1 (flap endonuclease 1), DNA polymerase delta/epsilon, PCNA, and LIG1. There are potentially two processes by which DNA gap filling occurs: short patch repair and long patch repair. Short patch repair involves the insertion of a single missing nucleotide. At some SSBs, gap filling might include displacing two or more nucleotides (displacement of up to 12 bases has been reported). FEN1 is an endonuclease that removes the displaced 5′-residues. Multiple DNA polymerases, including DNA polymerase beta, are involved in the repair of SSBs. The type of DNA polymerase that is recruited is dependent on the source and type of SSB.

In the fourth stage, a DNA ligase such as LIG1 (Ligase I) or LIG3 (Ligase III) catalyzes joining of the ends. Short patch repair uses Ligase III, while long patch repair uses Ligase I.

Sometimes, SSBR is coupled with replication, and may involve one or more of CtIP, MRN, ERCC1, and FEN1. SSBR is often cell-cycle dependent (see FIG. 16 ).

In some embodiments, HDR is promoted by down-regulating the SSBR pathway. For example, in some embodiments, a Cas9 molecule, e.g., a nickase, and a gRNA can induce a nick in a desired location during G1 or another phase of the cell cycle. A nick that is unrepaired in G1 will be converted into a DSB in S/G2 when a replication fork passes through the nicked area. In G2, the HDR machinery is active and can engage the break. Thus, an SSBR down-regulator can increase the likelihood that a break is repaired by HDR. A SSBR down-regulator can also increase the likelihood that a nick in G2 is repaired by HDR by preventing the cell from engaging the SSBR machinery during G2.

Accordingly, in some embodiments, the methods described herein involve down-regulating SSBR in order to promote HDR. For instance, the methods may involve modulating, e.g., inhibiting, a component (e.g., exactly one component, or one or more components, e.g., two or three components) of the SSBR pathway, e.g., a component of Table VI.13 or VI.1(F). More specifically, the methods may involve modulating, e.g., inhibiting, exactly one of, or one or more of a PARP, PARP1, PARP2, XRCC1, DNA polymerase beta (Pol beta), DNA polymerase delta, DNA polymerase epsilon, PCNA, LIG1, PNK, PNKP, APE1, APTX, APLF, TDP1, LIG3, FEN1, CtIP, MRN, and ERCC1. Up-regulation of HDR, in some embodiments, is achieved using siRNA directed against one or more of the components listed in Table VI.13 or VI.1(F). In some embodiments, a SSBR pathway is down regulated using an HDR-enhancing gRNA that targets a component of a SSBR pathway, e.g., one or more of the components listed in Table VI.13 or VI.1(F) (e.g., a PARP, PARP1, PARP2, XRCC1, DNA polymerase beta (Pol beta), DNA polymerase delta, DNA polymerase epsilon, PCNA, LIG1, PNK, PNKP, APE1, APTX, APLF, TDP1, LIG3, FEN1, CtIP, MRN, and ERCC1). In some embodiments, the HDR-enhancing gRNA is used in combination with an eiCas9 molecule. In some embodiments, the HDR-enhancing gRNA is used in combination with an eaCas9 molecule. In some embodiments, the HDR-enhancing gRNA is administered to the cell as a HDR-enhancing gRNA:Cas9 molecule complex.

In embodiments, one or more of HDR, alt-HR, anti-HR, NHEJ, MMEJ, SSA, MMR, NER, and BER are not substantially down-regulated, e.g., the only DNA damage repair pathway to be substantially down-regulated is the SSBR pathway.

TABLE VI.13 Factors that promote SSBR a PARP — PARP1 See Table VI.2 PARP2 See Table VI.2 PARG >sp|Q86W56|PARG_HUMAN Poly(ADP-ribose) glycohydrolase OS = Homo sapiens GN = PARG PE = 1 SV = 1 (SEQ ID NO: 326) MNAGPGCEPCTKRPRWGAATTSPAASDARSFPSRQRRVLDPKDAHVQFRVPPSSPACVPG RAGQHRGSATSLVFKQKTITSWMDTKGIKTAESESLDSKENNNTRIESMMSSVQKDNFYQ HNVEKLENVSQLSLDKSPTEKSTQYLNQHQTAAMCKWQNEGKHTEQLLESEPQTVTLVPE QFSNANIDRSPQNDDHSDTDSEENRDNQQFLTTVKLANAKQTTEDEQAREAKSHQKCSKS CDPGEDCASCQQDEIDVVPESPLSDVGSEDVGTGPKNDNKLTRQESCLGNSPPFEKESEP ESPMDVDNSKNSCQDSEADEETSPGFDEQEDGSSSQTANKPSRFQARDADIEFRKRYSTK GGEVRLHFQFEGGESRTGMNDLNAKLPGNISSLNVECRNSKQHGKKDSKITDHFMRLPKA EDRRKEQWETKHQRTERKIPKYVPPHLSPDKKWLGTPIEEMRRMPRCGIRLPLLRPSANH TVTIRVDLLRAGEVPKPFPTHYKDLWDNKHVKMPCSEQNLYPVEDENGERTAGSRWELIQ TALLNKFTRPQNLKDAILKYNVAYSKKWDFTALIDFWDKVLEEAEAQHLYQSILPDMVKI ALCLPNICTQPIPLLKQKMNHSITMSQEQIASLLANAFFCTFPRRNAKMKSEYSSYPDIN FNRLFEGRSSRKPEKLKTLFCYFRRVTEKKPTGLVTFTRQSLEDFPEWERCEKPLTRLHV TYEGTIEENGQGMLQVDFANRFVGGGVTSAGLVQEEIRFLINPELIISRLFTEVLDHNEC LIITGTEQYSEYTGYAETYRWSRSHEDGSERDDWQRRCTEIVAIDALHFRRYLDQFVPEK MRRELNKAYCGFLRPGVSSENLSAVATGNWGCGAFGGDARLKALIQILAAAAAERDVVYF TFGDSELMRDIYSMHIFLTERKLTVGDVYKLLLRYYNEECRNCSTPGPDIKLYPFIYHAV ESCAETADHSGQRTGT >sp|Q86W56-2|PARG_HUMAN Isoform 2 of Poly(ADP-ribose) glycohydrolase OS = Homo sapiens GN = PARG (SEQ ID NO: 327) MDTKGIKTAESESLDSKENNNTRIESMMSSVQKDNFYQHNVEKLENVSQLSLDKSPTEKS TQYLNQHQTAAMCKWQNEGKHTEQLLESEPQTVTLVPEQFSNANIDRSPQNDDHSDTDSE ENRDNQQFLTTVKLANAKQTTEDEQAREAKSHQKCSKSCDPGEDCASCQQDEIDVVPESP LSDVGSEDVGTGPKNDNKLTRQESCLGNSPPFEKESEPESPMDVDNSKNSCQDSEADEET SPGFDEQEDGSSSQTANKPSRFQARDADIEFRKRYSTKGGEVRLHFQFEGGESRTGMNDL NAKLPGNISSLNVECRNSKQHGKKDSKITDHFMRLPKAEDRRKEQWETKHQRTERKIPKY VPPHLSPDKKWLGTPIEEMRRMPRCGIRLPLLRPSANHTVTIRVDLLRAGEVPKPFPTHY KDLWDNKHVKMPCSEQNLYPVEDENGERTAGSRWELIQTALLNKFTRPQNLKDAILKYNV AYSKKWDFTALIDFWDKVLEEAEAQHLYQSILPDMVKIALCLPNICTQPIPLLKQKMNHS ITMSQEQIASLLANAFFCTFPRRNAKMKSEYSSYPDINFNRLFEGRSSRKPEKLKTLFCY FRRVTEKKPTGLVTFTRQSLEDFPEWERCEKPLTRLHVTYEGTIEENGQGMLQVDFANRF VGGGVTSAGLVQEEIRFLINPELIISRLFTEVLDHNECLIITGTEQYSEYTGYAETYRWS RSHEDGSERDDWQRRCTEIVAIDALHFRRYLDQFVPEKMRRELNKAYCGFLRPGVSSENL SAVATGNWGCGAFGGDARLKALIQILAAAAAERDVVYFTFGDSELMRDIYSMHIFLTERK LTVGDVYKLLLRYYNEECRNCSTPGPDIKLYPFIYHAVESCAETADHSGQRTGT >sp|Q86W56-3|PARG_HUMAN Isoform 3 of Poly(ADP-ribose) glycohydrolase OS = Homo sapiens GN = PARG (SEQ ID NO: 328) MMSSVQKDNFYQHNVEKLENVSQLSLDKSPTEKSTQYLNQHQTAAMCKWQNEGKHTEQLL ESEPQTVTLVPEQFSNANIDRSPQNDDHSDTDSEENRDNQQFLTTVKLANAKQTTEDEQA REAKSHQKCSKSCDPGEDCASCQQDEIDVVPESPLSDVGSEDVGTGPKNDNKLTRQESCL GNSPPFEKESEPESPMDVDNSKNSCQDSEADEETSPGFDEQEDGSSSQTANKPSRFQARD ADIEFRKRYSTKGGEVRLHFQFEGGESRTGMNDLNAKLPGNISSLNVECRNSKQHGKKDS KITDHFMRLPKAEDRRKEQWETKHQRTERKIPKYVPPHLSPDKKWLGTPIEEMRRMPRCG IRLPLLRPSANHTVTIRVDLLRAGEVPKPFPTHYKDLWDNKHVKMPCSEQNLYPVEDENG ERTAGSRWELIQTALLNKFTRPQNLKDAILKYNVAYSKKWDFTALIDFWDKVLEEAEAQH LYQSILPDMVKIALCLPNICTQPIPLLKQKMNHSITMSQEQIASLLANAFFCTFPRRNAK MKSEYSSYPDINFNRLFEGRSSRKPEKLKTLFCYFRRVTEKKPTGLVTFTRQSLEDFPEW ERCEKPLTRLHVTYEGTIEENGQGMLQVDFANRFVGGGVTSAGLVQEEIRFLINPELIIS RLFTEVLDHNECLIITGTEQYSEYTGYAETYRWSRSHEDGSERDDWQRRCTEIVAIDALH FRRYLDQFVPEKMRRELNKAYCGFLRPGVSSENLSAVATGNWGCGAFGGDARLKALIQIL AAAAAERDVVYFTFGDSELMRDIYSMHIFLTERKLTVGDVYKLLLRYYNEECRNCSTPGP DIKLYPFIYHAVESCAETADHSGQRTGT >sp|Q86W56-4|PARG_HUMAN Isoform 4 of Poly(ADP-ribose) glycohydrolase OS = Homo sapiens GN = PARG (SEQ ID NO: 329) MVQAGAEKDAQSISLRKEQWETKHQRTERKIPKYVPPHLSPDKKWLGTPIEEMRRMPRCG IRLPLLRPSANHTVTIWNGERTAGSRWELIQTALLNKFTRPQNLKDAILKYNVAYSKKWD FTALIDFWDKVLEEAEAQHLYQSILPDMVKIALCLPNICTQPIPLLKQKMNHSITMSQEQ IASLLANAFFCTFPRRNAKMKSEYSSYPDINFNRLFEGRSSRKPEKLKTLFCYFRRVTEK KPTGLVTFTRQSLEDFPEWERCEKPLTRLHVTYEGTIEENGQGMLQVDFANRFVGGGVTS AGLVQEEIRFLINPELIISRLFTEVLDHNECLIITGTEQYSEYTGYAETYRWSRSHEDGS ERDDWQRRCTEIVAIDALHFRRYLDQFVPEKMRRELNKAYCGFLRPGVSSENLSAVATGN WGCGAFGGDARLKALIQILAAAAAERDVVYFTFGDSELMRDIYSMHIFLTERKLTVGDVY KLLLRYYNEECRNCSTPGPDIKLYPFIYHAVESCAETADHSGQRTGT >sp|Q86W56-5|PARG_HUMAN Isoform 5 of Poly(ADP-ribose) glycohydrolase OS = Homo sapiens GN = PARG (SEQ ID NO: 330) MRRMPRCGIRLPLLRPSANHTVTIWNGERTAGSRWELIQTALLNKFTRPQNLKDAILKYN VAYSKKWDFTALIDFWDKVLEEAEAQHLYQSILPDMVKIALCLPNICTQPIPLLKQKMNH SITMSQEQIASLLANAFFCTFPRRNAKMKSEYSSYPDINFNRLFEGRSSRKPEKLKTLFC YFRRVTEKKPTGLVTFTRQSLEDFPEWERCEKPLTRLHVTYEGTIEENGQGMLQVDFANR FVGGGVTSAGLVQEEIRFLINPELIISRLFTEVLDHNECLIITGTEQYSEYTGYAETYRW SRSHEDGSERDDWQRRCTEIVAIDALHFRRYLDQFVPEKMRRELNKAYCGFLRPGVSSEN LSAVATGNWGCGAFGGDARLKALIQILAAAAAERDVVYFTFGDSELMRDIYSMHIFLTER KLTVGDVYKLLLRYYNEECRNCSTPGPDIKLYPFIYHAVESCAETADHSGQRTGT XRCC1 See Table VI.9 DNA polymerase MSLRSGGRRRADPGADGEASRDDGATSSVSALKRLERSQWTDKMDLRFGFERLKEPGEKTGWLINMHPTE beta ILDEDKRLGSAVDYYFIQDDGSRFKVALPYKPYFYIATRKGCEREVSSFLSKKFQGKIAKVETVPKEDLD LPNHLVGLKRNYIRLSFHTVEDLVKVRKEISPAVKKNREQDHASDAYTALLSSVLQRGGVITDEEETSKK IADQLDNIVDMREYDVPYHIRLSIDLKIHVAHWYNVRYRGNAFPVEITRRDDLVERPDPVVLAFDIETTK LPLKFPDAETDQIMMISYMIDGQGYLITNREIVSEDIEDFEFTPKPEYEGPFCVFNEPDEAHLIQRWFEH VQETKPTIMVTYNGDFFDWPFVEARAAVHGLSMQQEIGFQKDSQGEYKAPQCIHMDCLRWVKRDSYLPVG SHNLKAAAKAKLGYDPVELDPEDMCRMATEQPQTLATYSVSDAVATYYLYMKYVHPFIFALCTIIPMEPD EVLRKGSGTLCEALLMVQAFHANIIFPNKQEQEFNKLTDDGHVLDSETYVGGHVEALESGVFRSDIPCRF RMNPAAFDFLLQRVEKTLRHALEEEEKVPVEQVTNFEEVCDEIKSKLASLKDVPSRIECPLIYHLDVGAM YPNIILTNRLQPSAMVDEATCAACDFNKPGANCQRKMAWQWRGEFMPASRSEYHRIQHQLESEKFPPLFP EGPARAFHELSREEQAKYEKRRLADYCRKAYKKIHITKVEERLTTICQRENSFYVDTVRAFRDRRYEFKG LHKVWKKKLSAAVEVGDAAEVKRCKNMEVLYDSLQLAHKCILNSFYGYVMRKGARWYSMEMAGIVCFTGA NIITQARELIEQIGRPLELDTDGIWCVLPNSFPENFVFKTTNVKKPKVTISYPGAMLNIMVKEGFTNDQY QELAEPSSLTYVTRSENSIFFEVDGPYLAMILPASKEEGKKLKKRYAVFNEDGSLAELKGFEVKRRGELQ LIKIFQSSVFEAFLKGSTLEEVYGSVAKVADYWLDVLYSKAANMPDSELFELISENRSMSRKLEDYGEQK STSISTAKRLAEFLGDQMVKDAGLSCRYIISRKPEGSPVTERAIPLAIFQAEPTVRKHFLRKWLKSSSLQ DFDIRAILDWDYYIERLGSAIQKIITIPAALQQVKNPVPRVKHPDWLHKKLLEKNDVYKQKKISELFTLE GRRQVTMAEASEDSPRPSAPDMEDFGLVKLPHPAAPVTVKRKRVLWESQEESQDLTPTVPWQEILGQPPA LGTSQEEWLVWLRFHKKKWQLQARQRLARRKRQRLESAEGVLRPGAIRDGPATGLGSFLRRTARSILDLP WQIVQISETSQAGLFRLWALVGSDLHCIRLSIPRVFYVNQRVAKAEEGASYRKVNRVLPRSNMVYNLYEY SVPEDMYQEHINEINAELSAPDIEGVYETQVPLLFRALVHLGCVCVVNKQLVRHLSGWEAETFALEHLEM RSLAQFSYLEPGSIRHIYLYHHAQAHKALFGIFIPSQRRASVFVLDTVRSNQMPSLGALYSAEHGLLLEK VGPELLPPPKHTFEVRAETDLKTICRAIQRFLLAYKEERRGPTLIAVQSSWELKRLASEIPVLEEFPLVP ICVADKINYGVLDWQRHGARRMIRHYLNLDTCLSQAFEMSRYFHIPIGNLPEDISTFGSDLFFARHLQRH NHLLWLSPTARPDLGGKEADDNCLVMEFDDQATVEINSSGCYSTVCVELDLQNLAVNTILQSHHVNDMEG ADSMGISFDVIQQASLEDMITGGQAASAPASYDETALCSNTFRILKSMVVGWVKEITQYHNIYADNQVMH FYRWLRSPSSLLHDPALHRTLHNMMKKLFLQLIAEFKRLGSSVIYANFNRIILCTKKRRVEDAIAYVEYI TSSIHSKETFHSLTISFSRCWEFLLWMDPSNYGGIKGKVSSRIHCGLQDSQKAGGAEDEQENEDDEEERD GEEEEEAEESNVEDLLENNWNILQFLPQAASCQNYFLMIVSAYIVAVYHCMKDGLRRSAPGSTPVRRRGA SQLSQEAEGAVGALPGMITFSQDYVANELTQSFFTITQKIQKKVTGSRNSTELSEMFPVLPGSHLLLNNP ALEFIKYVCKVLSLDTNITNQVNKLNRDLLRLVDVGEFSEEAQFRDPCRSYVLPEVICRSCNFCRDLDLC KDSSFSEDGAVLPQWLCSNCQAPYDSSAIEMTLVEVLQKKLMAFTLQDLVCLKCRGVKETSMPVYCSCAG DFALTIHTQVFMEQIGIFRNIAQHYGMSYLLETLEWLLQKNPQLGH (SEQ ID NO: 331) DNA polymerase MFSEQAAQRAHTLLSPPSANNATFARVPVATYTNSSQPFRLGERSFSRQYAHIYATRLIQMRPFLENRAQ delta QHWGSGVGVKKLCELQPEEKCCVVGTLFKAMPLQPSILREVSEEHNLLPQPPRSKYIHPDDELVLEDELQ RIKLKGTIDVSKLVTGTVLAVFGSVRDDGKFLVEDYCFADLAPQKPAPPLDTDRFVLLVSGLGLGGGGGE SLLGTQLLVDVVTGQLGDEGEQCSAAHVSRVILAGNLLSHSTQSRDSINKAKYLTKKTQAASVEAVKMLD EILLQLSASVPVDVMPGEFDPTNYTLPQQPLHPCMFPLATAYSTLQLVTNPYQATIDGVRFLGTSGQNVS DIFRYSSMEDHLEILEWTLRVRHISPTAPDTLGCYPFYKTDPFIFPECPHVYFCGNTPSFGSKIIRGPED QTVLLVTVPDFSATQTACLVNLRSLACQPISFSGFGAEDDDLGGLGLGP (Pol delta2, sequence 1) (SEQ ID NO: 332) MGGAGARGLAGCGAPRVNLLGLGEAVWTKQVRSVAMFSEQAAQRAHTLLSPPSANNATFARVPVATYTNS SQPFRLGERSFSRQYAHIYATRLIQMRPFLENRAQQHWGSGVGVKKLCELQPEEKCCVVGTLFKAMPLQP SILREVSEEHNLLPQPPRSKYIHPDDELVLEDELQRIKLKGTIDVSKLVTGTVLAVFGSVRDDGKFLVED YCFADLAPQKPAPPLDTDRFVLLVSGLGLGGGGGESLLGTQLLVDVVTGQLGDEGEQCSAAHVSRVILAG NLLSHSTQSRDSINKAKYLTKKTQAASVEAVKMLDEILLQLSASVPVDVMPGEFDPTNYTLPQQPLHPCM FPLATAYSTLQLVTNPYQATIDGVRFLGTSGQNVSDIFRYSSMEDHLEILEWTLRVRHISPTAPDTLGCY PFYKTDPFIFPECPHVYFCGNTPSFGSKIIRGPEDQTVLLVTVPDFSATQTACLVNLRSLACQPISFSGF GAEDDDLGGLGLGP (Pol delta2, sequence 2) (SEQ ID NO: 333) MADQLYLENIDEFVTDQNKIVTYKWLSYTLGVHVNQAKQMLYDYVERKRKENSGAQLHVTYLVSGSLIQN GHSCHKVAVVREDKLEAVKSKLAVTASIHVYSIQKAMLKDSGPLFNTDYDILKSNLQNCSKFSAIQCAAA VPRAPAESSSSSKKFEQSHLHMSSETQANNELTTNGHGPPASKQVSQQPKGIMGMFASKAAAKTQETNKE TKTEAKEVTNASAAGNKAPGKGNMMSNFFGKAAMNKFKVNLDSEQAVKEEKIVEQPTVSVTEPKLATPAG LKKSSKKAEPVKVLQKEKKRGKRVALSDDETKETENMRKKRRRIKLPESDSSEDEVFPDSPGAYEAESPS PPPPPSPPLEPVPKTEPEPPSVKSSSGENKRKRKRVLKSKTYLDGEGCIVTEKVYESESCTDSEEELNMK TSSVHRPPAMTVKKEPREERKGPKKGTAALGKANRQVSITGFFQRK (Pol delta3) (SEQ ID NO: 334) DNA polymerase MSLRSGGRRRADPGADGEASRDDGATSSVSALKRLERSQWTDKMDLRFGFERLKEPGEKTGWLINMHPTE epsilon ILDEDKRLGSAVDYYFIQDDGSRFKVALPYKPYFYIATRKGCEREVSSFLSKKFQGKIAKVETVPKEDLD LPNHLVGLKRNYIRLSFHTVEDLVKVRKEISPAVKKNREQDHASDAYTALLSSVLQRGGVITDEEETSKK IADQLDNIVDMREYDVPYHIRLSIDLKIHVAHWYNVRYRGNAFPVEITRRDDLVERPDPVVLAFDIETTK LPLKFPDAETDQIMMISYMIDGQGYLITNREIVSEDIEDFEFTPKPEYEGPFCVFNEPDEAHLIQRWFEH VQETKPTIMVTYNGDFFDWPFVEARAAVHGLSMQQEIGFQKDSQGEYKAPQCIHMDCLRWVKRDSYLPVG SHNLKAAAKAKLGYDPVELDPEDMCRMATEQPQTLATYSVSDAVATYYLYMKYVHPFIFALCTIIPMEPD EVLRKGSGTLCEALLMVQAFHANIIFPNKQEQEFNKLTDDGHVLDSETYVGGHVEALESGVFRSDIPCRF RMNPAAFDFLLQRVEKTLRHALEEEEKVPVEQVTNFEEVCDEIKSKLASLKDVPSRIECPLIYHLDVGAM YPNIILTNRLQPSAMVDEATCAACDFNKPGANCQRKMAWQWRGEFMPASRSEYHRIQHQLESEKFPPLFP EGPARAFHELSREEQAKYEKRRLADYCRKAYKKIHITKVEERLTTICQRENSFYVDTVRAFRDRRYEFKG LHKVWKKKLSAAVEVGDAAEVKRCKNMEVLYDSLQLAHKCILNSFYGYVMRKGARWYSMEMAGIVCFTGA NIITQARELIEQIGRPLELDTDGIWCVLPNSFPENFVFKTTNVKKPKVTISYPGAMLNIMVKEGFTNDQY QELAEPSSLTYVTRSENSIFFEVDGPYLAMILPASKEEGKKLKKRYAVFNEDGSLAELKGFEVKRRGELQ LIKIFQSSVFEAFLKGSTLEEVYGSVAKVADYWLDVLYSKAANMPDSELFELISENRSMSRKLEDYGEQK STSISTAKRLAEFLGDQMVKDAGLSCRYIISRKPEGSPVTERAIPLAIFQAEPTVRKHFLRKWLKSSSLQ DFDIRAILDWDYYIERLGSAIQKIITIPAALQQVKNPVPRVKHPDWLHKKLLEKNDVYKQKKISELFTLE GRRQVTMAEASEDSPRPSAPDMEDFGLVKLPHPAAPVTVKRKRVLWESQEESQDLTPTVPWQEILGQPPA LGTSQEEWLVWLRFHKKKWQLQARQRLARRKRQRLESAEGVLRPGAIRDGPATGLGSFLRRTARSILDLP WQIVQISETSQAGLFRLWALVGSDLHCIRLSIPRVFYVNQRVAKAEEGASYRKVNRVLPRSNMVYNLYEY SVPEDMYQEHINEINAELSAPDIEGVYETQVPLLFRALVHLGCVCVVNKQLVRHLSGWEAETFALEHLEM RSLAQFSYLEPGSIRHIYLYHHAQAHKALFGIFIPSQRRASVFVLDTVRSNQMPSLGALYSAEHGLLLEK VGPELLPPPKHTFEVRAETDLKTICRAIQRFLLAYKEERRGPTLIAVQSSWELKRLASEIPVLEEFPLVP ICVADKINYGVLDWQRHGARRMIRHYLNLDTCLSQAFEMSRYFHIPIGNLPEDISTFGSDLFFARHLQRH NHLLWLSPTARPDLGGKEADDNCLVMEFDDQATVEINSSGCYSTVCVELDLQNLAVNTILQSHHVNDMEG ADSMGISFDVIQQASLEDMITGGQAASAPASYDETALCSNTFRILKSMVVGWVKEITQYHNIYADNQVMH FYRWLRSPSSLLHDPALHRTLHNMMKKLFLQLIAEFKRLGSSVIYANFNRIILCTKKRRVEDAIAYVEYI TSSIHSKETFHSLTISFSRCWEFLLWMDPSNYGGIKGKVSSRIHCGLQDSQKAGGAEDEQENEDDEEERD GEEEEEAEESNVEDLLENNWNILQFLPQAASCQNYFLMIVSAYIVAVYHCMKDGLRRSAPGSTPVRRRGA SQLSQEAEGAVGALPGMITFSQDYVANELTQSFFTITQKIQKKVTGSRNSTELSEMFPVLPGSHLLLNNP ALEFIKYVCKVLSLDTNITNQVNKLNRDLLRLVDVGEFSEEAQFRDPCRSYVLPEVICRSCNFCRDLDLC KDSSFSEDGAVLPQWLCSNCQAPYDSSAIEMTLVEVLQKKLMAFTLQDLVCLKCRGVKETSMPVYCSCAG DFALTIHTQVFMEQIGIFRNIAQHYGMSYLLETLEWLLQKNPQLGH (SEQ ID NO: 335) PCNA >sp|P12004|PCNA_HUMAN Proliferating cell nuclear antigen OS = Homo sapiens GN = PCNA PE = 1 SV = 1 MFEARLVQGSILKKVLEALKDLINEACWDISSSGVNLQSMDSSHVSLVQLTLRSEGFDTY RCDRNLAMGVNLTSMSKILKCAGNEDIITLRAEDNADTLALVFEAPNQEKVSDYEMKLMD LDVEQLGIPEQEYSCVVKMPSGEFARICRDLSHIGDAVVISCAKDGVKFSASGELGNGNI KLSQTSNVDKEEEAVTIEMNEPVQLTFALRYLNFFTKATPLSSTVTLSMSADVPLVVEYK IADMGHLKYYLAPKIEDEEGS (SEQ ID NO: 336 ) LIG1 See Table VI.9 PNK See Table VI.7 APE1 MPKRGKKGAVAEDGDELRTEPEAKKSKTAAKKNDKEAAGEGPALYEDPPDQKTSPSGKPA TLKICSWNVDGLRAWIKKKGLDWVKEEAPDILCLQETKCSENKLPAELQELPGLSHQYWS APSDKEGYSGVGLLSRQCPLKVSYGIGDEEHDQEGRVIVAEFDSFVLVTAYVPNAGRGLV RLEYRQRWDEAFRKFLKGLASRKPLVLCGDLNVAHEEIDLRNPKGNKKNAGFTPQERQGF GELLQAVPLADSFRHLYPNTPYAYTFWTYMMNARSKNVGWRLDYFLLSHSLLPALCDSKI RSKALGSDHCPITLYLAL (APE-1 CCDS 9550.1) (SEQ ID NO: 337) APTX MMRVCWLVRQDSRHQRIRLPHLEAVVIGRGPETKITDKKCSRQQVQLKAECNKGYVKVKQ VGVNPTSIDSVVIGKDQEVKLQPGQVLHMVNELYPYIVEFEEEAKNPGLETHRKRKRSGN SDSIERDAAQEAEAGTGLEPGSNSGQCSVPLKKGKDAPIKKESLGHWSQGLKISMQDPKM QVYKDEQVVVIKDKYPKARYHWLVLPWTSISSLKAVAREHLELLKHMHTVGEKVIVDFAG SSKLRFRLGYHAIPSMSHVHLHVISQDFDSPCLKNKKHWNSFNTEYFLESQAVIEMVQEA GRVTVRDGMPELLKLPLRCHECQQLLPSIPQLKEHLRKHWTQ (APTX Isoform 1 CCDS 47956.1) (SEQ ID NO: 338) MSNVNLSVSDFWRVMMRVCWLVRQDSRHQRIRLPHLEAVVIGRGPETKITDKKCSRQQEF EEEAKNPGLETHRKRKRSGNSDSIERDAAQEAEAGTGLEPGSNSGQCSVPLKKGKDAPIK KESLGHWSQGLKISMQDPKMQVYKDEQVVVIKDKYPKARYHWLVLPWTSISSLKAVAREH LELLKHMHTVGEKVIVDFAGSSKLRFRLGYHAIPSMSHVHLHVISQDFDSPCLKNKKHWN SFNTEYFLESQAVIEMVQEAGRVTVRDGMPELLKLPLRCHECQQLLPSIPQLKEHLRKHW TQ (APTX Isoform 2 CCDS 56568.1) (SEQ ID NO: 340) MMRVCWLVRQDSRHQRIRLPHLEAVVIGRGPETKITDKKCSRQQEFEEEAKNPGLETHRK RKRSGNSDSIERDAAQEAEAGTGLEPGSNSGQCSVPLKKGKDAPIKKESLGHWSQGLKIS MQDPKMQVYKDEQVVVIKDKYPKARYHWLVLPWTSISSLKAVAREHLELLKHMHTVGEKV IVDFAGSSKLRFRLGYHAIPSMSHVHLHVISQDFDSPCLKNKKHWNSFNTEYFLESQAVI EMVQEAGRVTVRDGMPELLKLPLRCHECQQLLPSIPQLKEHLRKHWTQ (APTX Isoform 3 CCDS 75827.1) (SEQ ID NO: 342) APLF MSGGFELQPRDGGPRVALAPGETVIGRGPLLGITDKRVSRRHAILEVAGGQLRIKPIHTN PCFYQSSEKSQLLPLKPNLWCYLNPGDSFSLLVDKYIFRILSIPSEVEMQCTLRNSQVLD EDNILNETPKSPVINLPHETTGASQLEGSTEIAKTQMTPTNSVSFLGENRDCNKQQPILA ERKRILPTWMLAEHLSDQNLSVPAISGGNVIQGSGKEEICKDKSQLNTTQQGRRQLISSG SSENTSAEQDTGEECKNTDQEESTISSKEMPQSFSAITLSNTEMNNIKTNAQRNKLPIEE LGKVSKHKIATKRTPHKEDEAMSCSENCSSAQGDSLQDESQGSHSESSSNPSNPETLHAK ATDSVLQGSEGNKVKRTSCMYGANCYRKNPVHFQHFSHPGDSDYGGVQIVGQDETDDRPE CPYGPSCYRKNPQHKIEYRHNTLPVRNVLDEDNDNVGQPNEYDLNDSFLDDEEEDYEPTD EDSDWEPGKEDEEKEDVEELLKEAKRFMKRK (APLF CCDS 1888.1) (SEQ ID NO: 343) TDP1 >tr|Q9BRS7|Q9BRS7_HUMAN TDP1 protein OS = Homo sapiens GN = TDP1 PE = 1 SV = 1 (SEQ ID NO: 347) MSQEGDYGRWTISSSDESEEEKPKPDKPSTSSLLCARQGAANEPRYTCSEAQKAAHKRKI SPVKFSNTDSVLPPKRQKSGSQEDLGWCLSSSDDELQPEMPQKQAEKVVIKKEKDISAPN DGTAQRTENHGAPACHRLKEEEDEYETSGEGQDIWDMLDKGNPFQFYLTRVSGVKPKYNS GALHIKDILSPLFGTLVSSAQFNYCFDVDWLVKQYPPEFRKKPILLVHGDKREAKAHLHA QAKPYENISLCQAKLDIAFGTHHTKMMLLLYEEGLRVVIHTSNLIHADWHQKTQGTHL LIG3 See Table VI.9 FEN1 See Table VI.2 CtIP See Table VI.2 MRN — ERCC1 See Table VI.9

More specifically, in some embodiments, the SSBR down-regulator is an inhibitor of a PARP such as AZD2281 (also called Olaparib and KU-0059436) or niraparib (produced by Tesero), BMN6′73 (produced by BioMarin Pharmaceutical), or rucaparib (produced by Clovis Oncology), an siRNA targeting XRCC1, or an agent Table VI.14, or any combination thereof. Inhibitors of some of the proteins of Table VI.13 above are also listed elsewhere in this specification.

TABLE VI.14 Down-regulators of SSBR Compounds Phase 1. PARP Olaparib (also known as AZD2281 and Phase III KU-0059436) Iniparib (also known as BSI-201) Phase III BMN 673 Phase III Rucaparib (also known as AG014699 and Phase II PF-01367338) Veliparib (also known as ABT-888) Phase II CEP 9722 Phase II INO-1001 Phase I/II MK 4827 Phase I BGB-290 Phase I E701 (also known as GPI21016) Phase I MP-124 Phase I LT-673 Preclinical NMS-P118 Preclinical XAV939 Preclinical 3-aminobenzamide Preclinical (highly selective against PARP-5) PARP1 siRNAs Accell Human PARP1 siRNA, sold by Dharmacon PARP1 Silencer, sold by Life Technologies PARP1 antibodies PARP1 antibody, sold by Proteintech Group Inc. PARP1 monoclonal antibody (M01), clone 3G4, sold by Abnova PARP2 siRNAs PARP2 Silencer, sold by Life Technologies Accell Human PARP2 siRNA, sold by Dharmacon PARP2 antibodies Anti-PARP-2, clone 4G8 antibody, sold by EMD Millipore PARP2 Antibody, sold by ProSci 2. XRCC1 siRNA XRCC1 Silencer, sold by Life Technologies Accell Human XRCC1 siRNA, sold by Dharmacon Commercially available siRNAs from Dharmacon or Ambion antibodies Anti-XRCC1 antibody, sold by Boster XRCC1 mouse monoclonal antibody, clone 2D8, sold by OriGene Technologies

VI.7 Mismatch Repair (MMR)

Cells contain three excision repair pathways: MMR, BER, and NER. The excision repair pathways have a common feature in that they typically recognize a lesion on one strand of the DNA, then exo/endonucleases remove the lesion and leave a 1-30 nucleotide gap that is subsequentially filled-in by DNA polymerase, and finally sealed by a ligase (see, e.g., Li (2008) CELL RESEARCH 18(1): 85-98). A brief description of the MMR is provided below.

Mismatch repair (MMR) operates on mispaired DNA bases. The MSH2/6 or MSH2/3 complexes both have ATPase activities that play an important role in mismatch recognition and the initiation of repair. MSH2/6 preferentially recognizes base-base mismatches and identifies mispairs of 1 or 2 nucleotides, while MSH2/3 preferentially recognizes larger insertion/deletion (ID) mispairs.

hMLH1 heterodimerizes with hPMS2 to form hMutLa, which possesses an ATPase activity, and is important for multiple steps of MMR. It possesses a PCNA/replication factor C (RFC)-dependent endonuclease activity which plays an important role in 3′ nick-directed MMR involving EXO1. (EXO1 is a participant in both HDR and MMR.) It regulates termination of mismatch-provoked excision. Ligase I is the relevant ligase for this pathway.

In some embodiments, the methods herein include down-regulating the MMR pathway in order to increase the frequency of successful genome editing. While not wishing to be bound by theory, MMR down-regulation could operate through the following mechanism. In some arrangements, genome editing will lead to a mismatch between the old and the new strand. Because MMR typically utilizes the original strand as the template, and therefore has a bias towards reverting back to the original sequence, down-regulation of MMR should enhance gene correction.

Accordingly, in some embodiments, the methods herein involve down-regulating the MMR pathway in order to promote HDR (e.g., HR, alt-HR or SSA). For instance, in some embodiments, the methods may involve modulating, e.g., inhibiting, a component (e.g., exactly one component, or one or more components, e.g., two or three components) of the MMR pathway, e.g., a component of Table VI.15 or VI.1(H). More specifically, in some embodiments, the methods may involve modulating, e.g., inhibiting, exactly one of, or one or more of EX01, MSH2, MSH3, MSH6, MLH1, PMS2, MLH3, DNA Polymerase delta, RPA, HMGB1, RFC, and DNA ligase I. Up-regulation of HDR, in some embodiments, is achieved using siRNA directed against one or more of the components listed in Table VI.15 or VI.1(H). In some embodiments, a MMR pathway is down regulated using an HDR-enhancing gRNA that targets a component of a MMR pathway, e.g., one or more of the components listed in Table VI.15 or VI.1(H) (e.g., one or more of EXO1, MSH2, MSH3, MSH6, MLH1, PMS2, MLH3, DNA Polymerase delta, RPA, HMGB1, RFC, and DNA ligase I). In some embodiments, the HDR-enhancing gRNA is used in combination with an eiCas9 molecule. In some embodiments, the HDR-enhancing gRNA is used in combination with an eaCas9 molecule. In some embodiments, the HDR-enhancing gRNA is administered to the cell as a HDR-enhancing gRNA:Cas9 molecule complex.

In some embodiments, one or more of HDR, alt-HR, anti-HR, NHEJ, MMEJ, SSA, SSBR, NER, and BER are not substantially down-regulated, e.g., in some embodiments the only DNA damage repair pathway to be substantially down-regulated is the MMR pathway.

TABLE VI.15 Factors involved in MMR. Factor Sequence EXO1 See Table VI.2 MSH2 >sp|P43246|MSH2_HUMAN DNA mismatch repair protein Msh2 OS = Homo sapiens GN = MSH2 PE = 1 SV = 1 (SEQ ID NO: 348) MAVQPKETLQLESAAEVGFVRFFQGMPEKPTTTVRLFDRGDFYTAHGEDALLAAREVFKT QGVIKYMGPAGAKNLQSVVLSKMNFESFVKDLLLVRQYRVEVYKNRAGNKASKENDWYLA YKASPGNLSQFEDILFGNNDMSASIGVVGVKMSAVDGQRQVGVGYVDSIQRKLGLCEFPD NDQFSNLEALLIQIGPKECVLPGGETAGDMGKLRQIIQRGGILITERKKADFSTKDIYQD LNRLLKGKKGEQMNSAVLPEMENQVAVSSLSAVIKFLELLSDDSNFGQFELTTFDFSQYM KLDIAAVRALNLFQGSVEDTTGSQSLAALLNKCKTPQGQRLVNQWIKQPLMDKNRIEERL NLVEAFVEDAELRQTLQEDLLRRFPDLNRLAKKFQRQAANLQDCYRLYQGINQLPNVIQA LEKHEGKHQKLLLAVFVTPLTDLRSDFSKFQEMIETTLDMDQVENHEFLVKPSFDPNLSE LREIMNDLEKKMQSTLISAARDLGLDPGKQIKLDSSAQFGYYFRVTCKEEKVLRNNKNFS TVDIQKNGVKFTNSKLTSLNEEYTKNKTEYEEAQDAIVKEIVNISSGYVEPMQTLNDVLA QLDAVVSFAHVSNGAPVPYVRPAILEKGQGRIILKASRHACVEVQDEIAFIPNDVYFEKD KQMFHIITGPNMGGKSTYIRQTGVIVLMAQIGCFVPCESAEVSIVDCILARVGAGDSQLK GVSTFMAEMLETASILRSATKDSLIIIDELGRGTSTYDGFGLAWAISEYIATKIGAFCMF ATHFHELTALANQIPTVNNLHVTALTTEETLTMLYQVKKGVCDQSFGIHVAELANFPKHV IECAKQKALELEEFQYIGESQGYDIMEPAAKKCYLEREQGEKIIQEFLSKVKQMPFTEMS EENITIKLKQLKAEVIAKNNSFVNEIISRIKVTT >sp|P43246-2|MSH2_HUMAN Isoform 2 of DNA mismatch repair protein Msh2 OS = Homo sapiens GN = MSH2 (SEQ ID NO: 349) MGPAGAKNLQSVVLSKMNFESFVKDLLLVRQYRVEVYKNRAGNKASKENDWYLAYKASPG NLSQFEDILFGNNDMSASIGVVGVKMSAVDGQRQVGVGYVDSIQRKLGLCEFPDNDQFSN LEALLIQIGPKECVLPGGETAGDMGKLRQIIQRGGILITERKKADFSTKDIYQDLNRLLK GKKGEQMNSAVLPEMENQVAVSSLSAVIKFLELLSDDSNFGQFELTTFDFSQYMKLDIAA VRALNLFQGSVEDTTGSQSLAALLNKCKTPQGQRLVNQWIKQPLMDKNRIEERLNLVEAF VEDAELRQTLQEDLLRRFPDLNRLAKKFQRQAANLQDCYRLYQGINQLPNVIQALEKHEG KHQKLLLAVFVTPLTDLRSDFSKFQEMIETTLDMDQVENHEFLVKPSFDPNLSELREIMN DLEKKMQSTLISAARDLGLDPGKQIKLDSSAQFGYYFRVTCKEEKVLRNNKNFSTVDIQK NGVKFTNSKLTSLNEEYTKNKTEYEEAQDAIVKEIVNISSGYVEPMQTLNDVLAQLDAVV SFAHVSNGAPVPYVRPAILEKGQGRIILKASRHACVEVQDEIAFIPNDVYFEKDKQMFHI ITGPNMGGKSTYIRQTGVIVLMAQIGCFVPCESAEVSIVDCILARVGAGDSQLKGVSTFM AEMLETASILRSATKDSLIIIDELGRGTSTYDGFGLAWAISEYIATKIGAFCMFATHFHE LTALANQIPTVNNLHVTALTTEETLTMLYQVKKGVCDQSFGIHVAELANFPKHVIECAKQ KALELEEFQYIGESQGYDIMEPAAKKCYLEREQGEKIIQEFLSKVKQMPFTEMSEENITI KLKQLKAEVIAKNNSFVNEIISRIKVTT MSH3 >sp|P20585|MSH3_HUMAN DNA mismatch repair protein Msh3 OS = Homo sapiens GN = MSH3 PE = 1 SV = 4 (SEQ ID NO: 350) MSRRKPASGGLAASSSAPARQAVLSRFFQSTGSLKSTSSSTGAADQVDPGAAAAAAAAAA AAPPAPPAPAFPPQLPPHIATEIDRRKKRPLENDGPVKKKVKKVQQKEGGSDLGMSGNSE PKKCLRTRNVSKSLEKLKEFCCDSALPQSRVQTESLQERFAVLPKCTDFDDISLLHAKNA VSSEDSKRQINQKDTTLFDLSQFGSSNTSHENLQKTASKSANKRSKSIYTPLELQYIEMK QQHKDAVLCVECGYKYRFFGEDAEIAARELNIYCHLDHNFMTASIPTHRLFVHVRRLVAK GYKVGVVKQTETAALKAIGDNRSSLFSRKLTALYTKSTLIGEDVNPLIKLDDAVNVDEIM TDTSTSYLLCISENKENVRDKKKGNIFIGIVGVQPATGEVVFDSFQDSASRSELETRMSS LQPVELLLPSALSEQTEALIHRATSVSVQDDRIRVERMDNIYFEYSHAFQAVTEFYAKDT VDIKGSQIISGIVNLEKPVICSLAAIIKYLKEFNLEKMLSKPENFKQLSSKMEFMTINGT TLRNLEILQNQTDMKTKGSLLWVLDHTKTSFGRRKLKKWVTQPLLKLREINARLDAVSEV LHSESSVFGQIENHLRKLPDIERGLCSIYHKKCSTQEFFLIVKTLYHLKSEFQAIIPAVN SHIQSDLLRTVILEIPELLSPVEHYLKILNEQAAKVGDKTELFKDLSDFPLIKKRKDEIQ GVIDEIRMHLQEIRKILKNPSAQYVTVSGQEFMIEIKNSAVSCIPTDWVKVGSTKAVSRF HSPFIVENYRHLNQLREQLVLDCSAEWLDFLEKFSEHYHSLCKAVHHLATVDCIFSLAKV AKQGDYCRPTVQEERKIVIKNGRHPVIDVLLGEQDQYVPNNTDLSEDSERVMIITGPNMG GKSSYIKQVALITIMAQIGSYVPAEEATIGIVDGIFTRMGAADNIYKGQSTFMEELTDTA EIIRKATSQSLVILDELGRGTSTHDGIAIAYATLEYFIRDVKSLTLFVTHYPPVCELEKN YSHQVGNYHMGFLVSEDESKLDPGAAEQVPDFVTFLYQIIRGIAARSYGLNVAKLADVPG EILKKAAHKSKELEGLINTKRKRLKYFAKLWIMHNAQDLQKWTEEFNMEETQTSLLH MSH6 >sp|P52701|MSH6_HUMAN DNA mismatch repair protein Msh6 OS = Homo sapiens GN = MSH6 PE = 1 SV = 2 (SEQ ID NO: 351) MSRQSTLYSFFPKSPALSDANKASARASREGGRAAAAPGASPSPGGDAAWSEAGPGPRPL ARSASPPKAKNLNGGLRRSVAPAAPTSCDFSPGDLVWAKMEGYPWWPCLVYNHPFDGIFI REKGKSVRVHVQFFDDSPIRGWVSKRLLKPYIGSKSKEAQKGGHFYSAKPEILRAMQRAD EALNKDKIKRLELAVCDEPSEPEEEEEMEVGTTYVTDKSEEDNEIESEEEVQPKTQGSRR SSRQIKKRRVISDSESDIGGSDVEFKPDTKEEGSSDEISSGVGDSESEGLNSPVKVARKR KRMVIGNGSLKRKSSRKETPSATKQATSISSETKNTLRAFSAPQNSESQAHVSGGGDDSS RPTVWYHETLEWLKEEKRRDEHRRRPDHPDFDASTLYVPEDFLNSCIPGMRKWWQIKSQN FDLVICYKVGKFYELYHMDALIGVSELGLVFMKGNWAHSGFPEIAFGRYSDSLVQKGYKV ARVEQTETPERMEARCRKMAHISKYDRVVRREICRIITKGTQTYSVLEGDPSENYSKYLL SLKEKEEDSSGHTRAYGVCFVDTSLGKFFIGQFSDDRHCSRFRILVAHYPPVQVLFEKGN LSKETKTILKSSLSCSLQEGLIPGSQFWDASKTLRILLEEEYFREKLSDGIGVMLPQVLK GMTSESDSIGLIPGEKSELALSALGGCVFYLKKCLIDQELLSMANFEEYIPLDSDIVSTT RSGAIFTKAYQRMVLDAVILNNLEIFLNGINGSTEGILLERVDTCHTPFGKRLLKQWLCA PLCNHYAINDRLDAIEDLMVVPDKISEVVELLKKLPDLERLLSKIHNVGSPLKSQNHPDS RAIMYEETTYSKKKIIDFLSALEGFKVMCKIIGIMEEVADGFKSKILKQVISLQTKNPEG RFPDLIVELNRWDTAFDHEKARKTGLITPKAGFDSDYDQALADIRENEQSLLEYLEKQRN RIGCRTIVYWGIGRNRYQLEIPENFTTRNLPEEYELKSIKKGCKRYWIKTIEKKLANLIN AEERRDVSLKDCMRRLFYNFDKNYKDWQSAVECIAVLDVLLCLANYSRGGDGPMCRPVIL LPEDIPPFLELKGSRHPCITKIFFGDDFIPNDILIGCEEEEQENGKAYCVLVTGPNMGGK STLMRQAGLLAVMAQMGCYVPAEVCRLIPIDRVFIRLGASDRIMSGESTFFVELSETASI LMHATAHSLVLVDELGRGTATFDGTAIANAVVKELAETIKCRTLFSTHYHSLVEDYSQNV AVRLGHMACMVENECEDPSQETITFLYKFIKGACPKSYGFNAARLANLPEEVIQKGHRKA REFEKMNQSLRLFREVCLASERSTVDAEAVHKLLTLIKEL MLH1 >sp|P40692|MLH1_HUMAN DNA mismatch repair protein Mlh1 OS = Homo sapiens GN = MLH1 PE = 1 SV = 1 (SEQ ID NO: 352) MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIVKEGGLKLIQ IQDNGTGIRKEDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTA DGKCAYRASYSDGKLKAPPKPCAGNQGTQIIVEDLEYNIATRRKALKNPSEEYGKILEVV GRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIRSIFGNAVSRELIEIGCEDKILAF KMNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIETVYAAYLPKNTHPFLYLSLEISP QNVDVNVHPIKHEVHFLHEESILERVQQHIESKLLGSNSSRMYFTQTLLPGLAGPSGEMV KSTISLISSSTSGSSDKVYAHQMVRIDSREQKLDAFLQPLSKPLSSQPQAIVIEDKIDIS SGRARQQDEEMLELPAPAEVAAKNQSLEGDTTKGTSEMSEKRGPTSSNPRKRHREDSDVE MVEDDSRKEMTAACTPRRRIINLTSVLSLQEEINEQGHEVLREMLHNHSFVGCVNPQWAL AQHQTKLYLLNITKLSEELFYQILIYDFANFGVLRLSEPAPLFDLAMLALDSPESGWTEE DGPKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFILRL ATEVNWDEEKECFESLSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWIVEHIV YKALRSHILPPKHFTEDGNILQLANLPDLYKVFERC >sp|P40692-2|MLH1_HUMAN Isoform 2 of DNA mismatch repair protein Mlh1 OS = Homo sapiens GN = MLH1 (SEQ ID NO: 353) MNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIETVYAAYLPKNTHPFLYLSLEISPQ NVDVNVHPTKHEVHFLHEESILERVQQHIESKLLGSNSSRMYFTQTLLPGLAGPSGEMVK STTSLTSSSTSGSSDKVYAHQMVRTDSREQKLDAFLQPLSKPLSSQPQAIVTEDKTDISS GRARQQDEEMLELPAPAEVAAKNQSLEGDTTKGTSEMSEKRGPTSSNPRKRHREDSDVEM VEDDSRKEMTAACTPRRRIINLTSVLSLQEEINEQGHEVLREMLHNHSFVGCVNPQWALA QHQTKLYLLNTTKLSEELFYQILIYDFANFGVLRLSEPAPLFDLAMLALDSPESGWTEED GPKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFILRLA TEVNWDEEKECFESLSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWTVEHIVY KALRSHILPPKHFTEDGNILQLANLPDLYKVFERC >sp|P40692-3|MLH1_HUMAN Isoform 3 of DNA mismatch repair protein Mlh1 OS = Homo sapiens GN = MLH1 (SEQ ID NO: 354) MAFEALASISHVAHVTITTKTADGKCAYRASYSDGKLKAPPKPCAGNQGTQITVEDLFYN IATRRKALKNPSEEYGKILEVVGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIRS IFGNAVSRELIEIGCEDKTLAFKMNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIET VYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILERVQQHIESKLLGSN SSRMYFTQILLPGLAGPSGEMVKSTISLTSSSTSGSSDKVYAHQMVRIDSREQKLDAFLQ PLSKPLSSQPQAIVTEDKTDISSGRARQQDEEMLELPAPAEVAAKNQSLEGDTTKGTSEM SEKRGPTSSNPRKRHREDSDVEMVEDDSRKEMTAACTPRRRIINLTSVLSLQEEINEQGH EVLREMLHNHSFVGCVNPQWALAQHQTKLYLLNTTKLSEELFYQILIYDFANFGVLRLSE PAPLFDLAMLALDSPESGWTEEDGPKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIG LPLLIDNYVPPLEGLPIFILRLATEVNWDEEKECFESLSKECAMFYSIRKQYISEESTLS GQQSEVPGSIPNSWKWTVEHIVYKALRSHILPPKHFTEDGNILQLANLPDLYKVFERC PMS2 >sp|P54278|PMS2_HUMAN Mismatch repair endonuclease PMS2 OS = Homo sapiens GN = PMS2 PE = 1 SV = 2 (SEQ ID NO: 355) MERAESSSTEPAKAIKPIDRKSVHQICSGQVVLSLSTAVKELVENSLDAGATNIDLKLKD YGVDLIEVSDNGCGVEEENFEGLTLKHHTSKIQEFADLTQVETFGFRGEALSSLCALSDV TISTCHASAKVGTRLMFDHNGKIIQKTPYPRPRGTTVSVQQLFSTLPVRHKEFQRNIKKE YAKMVQVLHAYCIISAGIRVSCINQLGQGKRQPVVCIGGSPSIKENIGSVFGQKQLQSLI PFVQLPPSDSVCEEYGLSCSDALHNLFYISGFISQCTHGVGRSSTDRQFFFINRRPCDPA KVCRLVNEVYHMYNRHQYPFVVLNISVDSECVDINVTPDKRQILLQEEKLLLAVLKTSLI GMFDSDVNKLNVSQQPLLDVEGNLIKMHAADLEKPMVEKQDQSPSLRTGEEKKDVSISRL REAFSLRHTTENKPHSPKTPEPRRSPLGQKRGMLSSSTSGAISDKGVLRPQKEAVSSSHG PSDPTDRAEVEKDSGHGSTSVDSEGFSIPDTGSHCSSEYAASSPGDRGSQEHVDSQEKAP KTDDSFSDVDCHSNQEDTGCKFRVLPQPTNLATPNTKRFKKEEILSSSDICQKLVNTQDM SASQVDVAVKINKKVVPLDFSMSSLAKRIKQLHHEAQQSEGEQNYRKFRAKICPGENQAA EDELRKEISKTMFAEMETIGQFNLGFIITKLNEDIFIVDQHATDEKYNFEMLQQHTVLQG QRLIAPQTLNLTAVNEAVLIENLEIFRKNGFDEVIDENAPVTERAKLISLPTSKNWTFGP QDVDELIFMLSDSPGVMCRPSRVKQMFASRACRKSVMIGTALNTSEMKKLITHMGEMDHP WNCPHGRPTMRHIANLGVISQN >sp|P54278-2|PMS2_HUMAN Isoform 2 of Mismatch repair endonuclease PMS2 OS = Homo sapiens GN = PMS2 (SEQ ID NO: 356) MERAESSSTEPAKAIKPIDRKSVHQICSGQVVLSLSTAVKELVENSLDAGATNIDLKLKD YGVDLIEVSDNGCGVEEENFEGLTLKHHTSKIQEFADLTQVETFGFRGEALSSLCALSDV TISTCHASAKVGTRLMFDHNGKIIQKTPYPRPRGTTVSVQQLFSTLPVRHKEFQRNIKKE YAKMVQVLHAYCIISAGIRVSCINQLGQGKRQPVVCIGGSPSIKENIGSVFGQKQLQSLI PFVQLPPSDSVCEEYGLSCSDALHNLFYKTMFAEMETIGQFNLGFIITKLNEDIFIVDQH ATDEKYNFEMLQQHTVLQGQRLIAPQTLNLTAVNEAVLIENLEIFRKNGFDEVIDENAPV TERAKLISLPTSKNWTFGPQDVDELIFMLSDSPGVMCRPSRVKQMFASRACRKSVMIGTA LNTSEMKKLITHMGEMDHPWNCPHGRPTMRHIANLGVISQN >sp|P54278-3|PMS2_HUMAN Isoform 3 of Mismatch repair endonuclease PMS2 OS = Homo sapiens GN = PMS2 (SEQ ID NO: 357) MERAESSSTEPAKAIKPIDRKSVHQICSGQVVLSLSTAVKELVENSLDAGATNIDLKLKD YGVDLIEVSDNGCGVEEENFEGLTLKHHTSKIQEFADLTQVETFGFRGEALSSLCALSDV TISTCHASAKVGTRLMFDHNGKIIQKTPYPRPRGTTVSVQQLFSTLPVRHKEFQRNIKKE YAKMVQVLHAYCIISAGIRVSCINQLGQGKRQPVVCIGGSPSIKENIGSVFGQKQLQSLI PFVQLPPSDSVCEEYGLSCSDALHNLFYISGFISQCTHGVGRSSTDRQFFFINRRPCDPA KVCRLVNEVYHMYNRHQYPFVVLNISVDSECVDINVTPDKRQILLQEEKLLLAVLKTSLI GMFDSDVNKLNVSQQPLLDVEGNLIKMHAADLEKPMVEKQDQSPSLRTGEEKKDVSISRL REAFSLRHTTENKPHSPKTPEPRRSPLGQKRGMLSSSTSGAISDKGVLRPQKEAVSSSHG PSDPTDRAEVEKDSGHGSTSVDSEGFSIPDTGSHCSSEYAASSPGDRGSQEHVDSQEKAP KTDDSFSDVDCHSNQEDTGLKTGPSDPRTSMN >sp|P54278-4|PMS2_HUMAN Isoform 4 of Mismatch repair endonuclease PMS2 OS = Homo sapiens GN = PMS2 (SEQ ID NO: 358) MERAESSSTEPAKAIKPIDRKSVHQICSGQVVLSLSTAVKELVENSLDAGATNIDLKLKD YGVDLIEVSDNGCGVEEENFEGLTLKHHTSKIQEFADLTQVETFGFRGEALSSLCALSDV TISTCHASAKVGTRLMFDHNGKIIQKTPYPRPRGTTVSVQQLFSTLPVRHKEFQRNIKKQ ASV MLH3 >sp|Q9UHC1|MLH3_HUMAN DNA mismatch repair protein Mlh3 OS = Homo sapiens GN = MLH3 PE = 1 SV = 3 (SEQ ID NO: 359) MIKCLSVEVQAKLRSGLAISSLGQCVEELALNSIDAEAKCVAVRVNMETFQVQVIDNGFG MGSDDVEKVGNRYFTSKCHSVQDLENPRFYGFRGEALANIADMASAVEISSKKNRTMKTF VKLFQSGKALKACEADVTRASAGTTVTVYNLFYQLPVRRKCMDPRLEFEKVRQRIEALSL MHPSISFSLRNDVSGSMVLQLPKTKDVCSRFCQIYGLGKSQKLREISFKYKEFELSGYIS SEAHYNKNMQFLFVNKRLVLRTKLHKLIDFLLRKESIICKPKNGPTSRQMNSSLRHRSTP ELYGIYVINVQCQFCEYDVCMEPAKTLIEFQNWDTLLFCIQEGVKMFLKQEKLFVELSGE DIKEFSEDNGFSLFDATLQKRVTSDERSNFQEACNNILDSYEMFNLQSKAVKRKTTAENV NTQSSRDSEATRKNTNDAFLYIYESGGPGHSKMTEPSLQNKDSSCSESKMLEQETIVASE AGENEKHKKSFLEHSSLENPCGTSLEMFLSPFQTPCHFEESGQDLEIWKESTTVNGMAAN ILKNNRIQNQPKRFKDATEVGCQPLPFATTLWGVHSAQTEKEKKKESSNCGRRNVFSYGR VKLCSTGFITHVVQNEKTKSTETEHSFKNYVRPGPTRAQETFGNRTRHSVETPDIKDLAS TLSKESGQLPNKKNCRTNISYGLENEPTATYTMFSAFQEGSKKSQTDCILSDTSPSFPWY RHVSNDSRKTDKLIGFSKPIVRKKLSLSSQLGSLEKFKRQYGKVENPLDTEVEESNGVTT NLSLQVEPDILLKDKNRLENSDVCKITTMEHSDSDSSCQPASHILNSEKFPFSKDEDCLE QQMPSLRESPMTLKELSLFNRKPLDLEKSSESLASKLSRLKGSERETQTMGMMSRFNELP NSDSSRKDSKLCSVLTQDFCMLFNNKHEKTENGVIPTSDSATQDNSFNKNSKTHSNSNTT ENCVISETPLVLPYNNSKVTGKDSDVLIRASEQQIGSLDSPSGMLMNPVEDATGDQNGIC FQSEESKARACSETEESNTCCSDWQRHFDVALGRMVYVNKMTGLSTFIAPTEDIQAACTK DLTTVAVDVVLENGSQYRCQPFRSDLVLPFLPRARAERTVMRQDNRDTVDDTVSSESLQS LFSEWDNPVFARYPEVAVDVSSGQAESLAVKIHNILYPYRFTKGMIHSMQVLQQVDNKFI ACLMSTKTEENGEAGGNLLVLVDQHAAHERIRLEQLIIDSYEKQQAQGSGRKKLLSSTLI PPLEITVTEEQRRLLWCYHKNLEDLGLEFVFPDTSDSLVLVGKVPLCFVEREANELRRGR STVTKSIVEEFIREQLELLQTTGGIQGTLPLTVQKVLASQACHGAIKFNDGLSLQESCRL IEALSSCQLPFQCAHGRPSMLPLADIDHLEQEKQIKPNLTKLRKMAQAWRLFGKAECDTR QSLQQSMPPCEPP >sp|Q9UHC1-2|MLH3_HUMAN Isoform 2 of DNA mismatch repair protein Mlh3 OS = Homo sapiens GN = MLH3 (SEQ ID NO: 360) MIKCLSVEVQAKLRSGLAISSLGQCVEELALNSIDAEAKCVAVRVNMETFQVQVIDNGFG MGSDDVEKVGNRYFTSKCHSVQDLENPRFYGFRGEALANIADMASAVEISSKKNRTMKTF VKLFQSGKALKACEADVTRASAGTTVTVYNLFYQLPVRRKCMDPRLEFEKVRQRIEALSL MHPSISFSLRNDVSGSMVLQLPKTKDVCSRFCQIYGLGKSQKLREISFKYKEFELSGYIS SEAHYNKNMQFLFVNKRLVLRTKLHKLIDFLLRKESIICKPKNGPTSRQMNSSLRHRSTP ELYGIYVINVQCQFCEYDVCMEPAKTLIEFQNWDTLLFCIQEGVKMFLKQEKLFVELSGE DIKEFSEDNGFSLFDATLQKRVTSDERSNFQEACNNILDSYEMFNLQSKAVKRKTTAENV NTQSSRDSEATRKNTNDAFLYIYESGGPGHSKMTEPSLQNKDSSCSESKMLEQETIVASE AGENEKHKKSFLEHSSLENPCGTSLEMFLSPFQTPCHFEESGQDLEIWKESTTVNGMAAN ILKNNRIQNQPKRFKDATEVGCQPLPFATTLWGVHSAQTEKEKKKESSNCGRRNVFSYGR VKLCSTGFITHVVQNEKTKSTETEHSFKNYVRPGPTRAQETFGNRTRHSVETPDIKDLAS TLSKESGQLPNKKNCRTNISYGLENEPTATYTMFSAFQEGSKKSQTDCILSDTSPSFPWY RHVSNDSRKTDKLIGFSKPIVRKKLSLSSQLGSLEKFKRQYGKVENPLDTEVEESNGVTT NLSLQVEPDILLKDKNRLENSDVCKITTMEHSDSDSSCQPASHILNSEKFPFSKDEDCLE QQMPSLRESPMTLKELSLFNRKPLDLEKSSESLASKLSRLKGSERETQTMGMMSRFNELP NSDSSRKDSKLCSVLTQDFCMLFNNKHEKTENGVIPTSDSATQDNSFNKNSKTHSNSNTT ENCVISETPLVLPYNNSKVTGKDSDVLIRASEQQIGSLDSPSGMLMNPVEDATGDQNGIC FQSEESKARACSETEESNTCCSDWQRHFDVALGRMVYVNKMTGLSTFIAPTEDIQAACTK DLTTVAVDVVLENGSQYRCQPFRSDLVLPFLPRARAERTVMRQDNRDTVDDTVSSESLQS LFSEWDNPVFARYPEVAVDVSSGQAESLAVKIHNILYPYRFTKGMIHSMQVLQQVDNKFI ACLMSTKTEENGEADSYEKQQAQGSGRKKLLSSTLIPPLEITVTEEQRRLLWCYHKNLED LGLEFVFPDTSDSLVLVGKVPLCFVEREANELRRGRSTVTKSIVEEFIREQLELLQTTGG IQGTLPLTVQKVLASQACHGAIKFNDGLSLQESCRLIEALSSCQLPFQCAHGRPSMLPLA DIDHLEQEKQIKPNLTKLRKMAQAWRLFGKAECDTRQSLQQSMPPCEPP DNA Polymerased See Table VI.13 elta RPA — HMGB1 >sp|P09429|HMGB1_HUMAN High mobility group protein B1 OS = Homo sapiens GN = HMGB1 PE = 1 SV = 3 (SEQ ID NO: 361) MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKF EDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRPPSAFFLFCSEYRPKIKGEHPGL SIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVVKAEK SKKKKEEEEDEEDEEDEEEEEDEEDEDEEEDDDDE >tr|Q5T7C4|Q5T7C4_HUMAN High mobility group protein B1 OS = Homo sapiens GN = HMGB1 PE = 1 SV = 1 (SEQ ID NO: 362) MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKF EDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRPPSAFFLFCSEYRPKIKGEHPGL SIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKF RFC >sp|P35251|RFC1_HUMAN Replication factor C subunit 1 OS = Homo sapiens GN = RFC1 PE = 1 SV = 4 (SEQ ID NO: 363) MDIRKFFGVIPSGKKLVSETVKKNEKTKSDEETLKAKKGIKEIKVNSSRKEDDFKQKQPS KKKRITYDSDSESEETLQVKNAKKPPEKLPVSSKPGKISRQDPVTYISETDEEDDFMCKK AASKSKENGRSTNSHLGTSNMKKNEENTKTKNKPLSPIKLTPTSVLDYFGTGSVQRSNKK MVASKRKELSQNTDESGLNDEATAKQLQLDEDAELERQLHEDEEFARTLAMLDEEPKTKK ARKDTEAGETFSSVQANLSKAEKHKYPHKVKTAQVSDERKSYSPRKQSKYESSKESQQHS KSSADKIGEVSSPKASSKLAIMKRKEESSYKEIEPVASKRKENAIKLKGETKTPKKTKSS PAKKESVSPEDSEKKRTNYQAYRSYLNREGPKALGSKEIPKGAENCLEGLIFVITGVLES IERDEAKSLIERYGGKVTGNVSKKTNYLVMGRDSGQSKSDKAAALGTKIIDEDGLLNLIR TMPGKKSKYEIAVETEMKKESKLERTPQKNVQGKRKISPSKKESESKKSRPTSKRDSLAK TIKKETDVFWKSLDFKEQVAEETSGDSKARNLADDSSENKVENLLWVDKYKPTSLKTIIG QQGDQSCANKLLRWLRNWQKSSSEDKKHAAKFGKFSGKDDGSSFKAALLSGPPGVGKTTT ASLVCQELGYSYVELNASDTRSKSSLKAIVAESLNNTSIKGFYSNGAASSVSTKHALIMD EVDGMAGNEDRGGIQELIGLIKHTKIPIICMCNDRNHPKIRSLVHYCFDLRFQRPRVEQI KGAMMSIAFKEGLKIPPPAMNEHILGANQDIRQVLHNLSMWCARSKALTYDQAKADSHRA KKDIKMGPFDVARKVFAAGEETAHMSLVDKSDLFFHDYSIAPLFVQENYIHVKPVAAGGD MKKHLMLLSRAADSICDGDLVDSQIRSKQNWSLLPAQAIYASVLPGELMRGYMTQFPTFP SWLGKHSSTGKHDRIVQDLALHMSLRTYSSKRTVNMDYLSLLRDALVQPLTSQGVDGVQD VVALMDTYYLMKEDFENIMEISSWGGKPSPFSKLDPKVKAAFTRAYNKEAHLTPYSLQAI KASRHSTSPSLDSEYNEELNEDDSQSDEKDQDAIETDAMIKKKTKSSKPSKPEKDKEPRK GKGKSSKK >sp|P35251-2|RFC1_HUMAN Isoform 2 of Replication factor C subunit 1 OS = Homo sapiens GN = RFC1 (SEQ ID NO: 364) MDIRKFFGVIPSGKKLVSETVKKNEKTKSDEETLKAKKGIKEIKVNSSRKEDDFKQKQPS KKKRIIYDSDSESEETLQVKNAKKPPEKLPVSSKPGKISRQDPVIYISETDEEDDFMCKK AASKSKENGRSINSHLGTSNMKKNEENTKIKNKPLSPIKLIPTSVLDYFGIGSVQRSNKK MVASKRKELSQNTDESGLNDEAIAKQLQLDEDAELERQLHEDEEFARTLAMLDEEPKIKK ARKDTEAGETFSSVQANLSKAEKHKYPHKVKTAQVSDERKSYSPRKQSKYESSKESQQHS KSSADKIGEVSSPKASSKLAIMKRKEESSYKEIEPVASKRKENAIKLKGETKIPKKIKSS PAKKESVSPEDSEKKRTNYQAYRSYLNREGPKALGSKEIPKGAENCLEGLIFVITGVLES IERDEAKSLIERYGGKVIGNVSKKTNYLVMGRDSGQSKSDKAAALGTKIIDEDGLLNLIR IMPGKKSKYEIAVETEMKKESKLERTPQKNVQGKRKISPSKKESESKKSRPTSKRDSLAK TIKKETDVFWKSLDFKEQVAEETSGDSKARNLADDSSENKVENLLWVDKYKPISLKTIIG QQGDQSCANKLLRWLRNWQKSSSEDKKHAKFGKFSGKDDGSSFKAALLSGPPGVGKTTTA SLVCQELGYSYVELNASDIRSKSSLKAIVAESLNNTSIKGFYSNGAASSVSTKHALIMDE VDGMAGNEDRGGIQELIGLIKHTKIPIICMCNDRNHPKIRSLVHYCFDLRFQRPRVEQIK GAMMSIAFKEGLKIPPPAMNEIILGANQDIRQVLHNLSMWCARSKALTYDQAKADSHRAK KDIKMGPFDVARKVFAAGEETAHMSLVDKSDLFFHDYSIAPLFVQENYIHVKPVAAGGDM KKHLMLLSRAADSICDGDLVDSQIRSKQNWSLLPAQAIYASVLPGELMRGYMTQFPTFPS WLGKHSSIGKHDRIVQDLALHMSLRTYSSKRIVNMDYLSLLRDALVQPLISQGVDGVQDV VALMDTYYLMKEDFENIMEISSWGGKPSPFSKLDPKVKAAFTRAYNKEAHLTPYSLQAIK ASRHSTSPSLDSEYNEELNEDDSQSDEKDQDAIETDAMIKKKIKSSKPSKPEKDKEPRKG KGKSSKK DNA ligase I VI.9

More specifically, in some embodiments, the down-regulator of the MMR pathway may be an inhibitor of (e.g., an siRNA against) one or more factors of Table VI.15, a down-regulator listed in Table VI.16, or any combination thereof. Inhibitors of some of the proteins of Table VI.15 above are listed elsewhere in this specification.

TABLE VI.16 Down-regulators of MMR 1. Msh2 siRNA Commercially available from Dharmacon or Ambion 2. Msh3 siRNA Commercially available from Dharmacon or Ambion 3. Msh6 siRNA Commercially available from Dharmacon or Ambion 4. Mlh1 siRNA Commercially available from Dharmacon or Ambion 5. Pms2 siRNA Commercially available from Dharmacon or Ambion 6. Compounds that down-regulate MMR Cadmium (Cd²⁺)

VI.8 Base Excision Repair (BER)

The base excision repair (BER) pathway is active throughout the cell cycle; it is responsible primarily for removing small, non-helix-distorting base lesions from the genome. In contrast, the related Nucleotide Excision Repair (NER) pathway (discussed below) repairs bulky helix-distorting lesions (see, e.g., Caldecott (2008) NAT. REV. GENET. 9: 619-31. A brief description of the BER is provided below.

Upon DNA base damage, BER is initiated and the process can be simplified into five major steps: (a) removal of the damaged DNA base; (b) incision of the subsequent a basic site; (c) clean-up of the DNA ends; (d) insertion of the correct nucleotide into the repair gap; and (e) ligation of the remaining nick in the DNA backbone. These last steps are similar to the SSBR pathway.

In the first step, a damage-specific DNA glycosylase excises the damaged base through cleavage of the N-glycosidic bond linking the base to the sugar phosphate backbone. Then AP endonuclease-1 (APE1) or bifunctional DNA glycosylases with an associated lyase activity incise the phosphodiester backbone to create a DNA single strand break (SSB). The third step of BER involves procession (i.e., cleaning-up) of the DNA ends. The fourth step in BER is conducted by DNA polymerase beta (Pol beta) which adds a new complementary nucleotide to the repair gap. In the final step, XRCC1/Ligase III seal the remaining nick in the DNA backbone. This completes the short-patch BER pathway through which the majority (˜80%) of damaged DNA bases are repaired. However, if the 5′-ends in step 3 are resistant to end-processing activity, following one nucleotide insertion by DNA polymerase beta, a polymerase switch to the replicative DNA polymerases delta/epsilon occurs, which then add ˜2-8 more nucleotides into the DNA repair gap. This creates a 5′-flap structure, which is recognized and excised by flap endonuclease-1 (FEN-1) in association with the processivity factor proliferating cell nuclear antigen (PCNA). DNA ligase I then seals the remaining nick in the DNA backbone and completes long-patch BER. Additional factors are listed in Table VI.17.

In some embodiments, the methods described herein involve down-regulating the BER pathway in order to promote HDR (e.g., HR, alt-HR or SSA). For instance, in some embodiments, the methods may involve modulating, e.g., inhibiting, a component (e.g., exactly one component, or one or more components, e.g., two or three components) of the BER pathway, e.g., a component of Table VI.17. More specifically, in some embodiments, the methods may involve modulating, e.g., inhibiting, exactly one of, or one or more of DNA glycosylase, APE1, Pol beta, Pol delta, Pol epsilon, XRCC1, Ligase III, FEN-1, PCNA, RECQL4, WRN, MYH, PNKP, and APTX. Up-regulation of HDR, in some embodiments, is achieved using siRNA directed against one or more of the components listed in Table VI.17. In some embodiments, a BER pathway is down regulated using an HDR-enhancing gRNA that targets a component of a BER pathway, e.g., one or more of the components listed in Table VI.17 (e.g., one or more of DNA glycosylase, APE1, Pol beta, Pol delta, Pol epsilon, XRCC1, Ligase III, FEN-1, PCNA, RECQL4, WRN, MYH, PNKP, and APTX). In some embodiments, the HDR-enhancing gRNA is used in combination with an eiCas9 molecule. In some embodiments, the HDR-enhancing gRNA is used in combination with an eaCas9 molecule. In some embodiments, the HDR-enhancing gRNA is administered to the cell as a HDR-enhancing gRNA:Cas9 molecule complex.

In some embodiments, one or more of HDR, alt-HR, anti-HR, NHEJ, MMEJ, SSA, SSBR, NER, and MMR are not substantially down-regulated, e.g., in some embodiments the only DNA damage repair pathway to be substantially down-regulated is the BER pathway.

TABLE VI.17 Factors Involved in BER Factor Sequence DNA glycosylase — APE1 See Table VI.13 Pol beta See Table VI.13 Pol delta See Table VI.13 Pol epsilon See Table VI.13 XRCC1 See Table VI.13 Ligase III See Table VI.9 FEN-1 See Table VI.2 PCNA See Table VI.13 RECQL4 MERLRDVRERLQAWERAFRRQRGRRPSQDDVEAAPEETRALYREYRTLKRTTGQAGGGLR SSESLPAAAEEAPEPRCWGPHLNRAATKSPQSTPGRSRQGSVPDYGQRLKANLKGTLQAG PALGRRPWPLGRASSKASTPKPPGTGPVPSFAEKVSDEPPQLPEPQPRPGRLQHLQASLS QRLGSLDPGWLQRCHSEVPDFLGAPKACRPDLGSEESQLLIPGESAVLGPGAGSQGPEAS AFQEVSIRVGSPQPSSSGGEKRRWNEEPWESPAQVQQESSQAGPPSEGAGAVAVEEDPPG EPVQAQPPQPCSSPSNPRYHGLSPSSQARAGKAEGTAPLHIFPRLARHDRGNYVRLNMKQ KHYVRGRALRSRLLRKQAWKQKWRKKGECFGGGGATVTTKESCFLNEQFDHWAAQCPRPA SEEDTDAVGPEPLVPSPQPVPEVPSLDPTVLPLYSLGPSGQLAETPAEVFQALEQLGHQA FRPGQERAVMRILSGISTLLVLPTGAGKSLCYQLPALLYSRRSPCLTLVVSPLLSLMDDQ VSGLPPCLKAACIHSGMTRKQRESVLQKIRAAQVHVLMLTPEALVGAGGLPPAAQLPPVA FACIDEAHCLSQWSHNFRPCYLRVCKVLRERMGVHCFLGLTATATRRTASDVAQHLAVAE EPDLHGPAPVPTNLHLSVSMDRDTDQALLTLLQGKRFQNLDSIIIYCNRREDTERIAALL RTCLHAAWVPGSGGRAPKTTAEAYHAGMCSRERRRVQRAFMQGQLRVVVATVAFGMGLDR PDVRAVLHLGLPPSFESYVQAVGRAGRDGQPAHCHLFLQPQGEDLRELRRHVHADSTDFL AVKRLVQRVFPACTCTCTRPPSEQEGAVGGERPVPKYPPQEAEQLSHQAAPGPRRVCMGH ERALPIQLTVQALDMPEEAIETLLCYLELHPHHWLELLATTYTHCRLNCPGGPAQLQALA HRCPPLAVCLAQQLPEDPGQGSSSVEFDMVKLVDSMGWELASVRRALCQLQWDHEPRTGV RRGTGVLVEFSELAFHLRSPGDLTAEEKDQICDFLYGRVQARERQALARLRRTFQAFHSV AFPSCGPCLEQQDEERSTRLKDLLGRYFEEEEGQEPGGMEDAQGPEPGQARLQDWEDQVR CDIRQFLSLRPEEKFSSRAVARIFHGIGSPCYPAQVYGQDRRFWRKYLHLSFHALVGLAT EELLQVAR (RECQL4 CCDS 75804.1) (SEQ ID NO: 365) WRN See Table VI.2 MYH MTPLVSRLSRLWAIMRKPRAAVGSGHRKQAASQEGRQKHAKNNSQAKPSACDGLARQPEEVVLQASVSSY HLFRDVAEVTAFRGSLLSWYDQEKRDLPWRRRAEDEMDLDRRAYAVWVSEVMLQQTQVATVINYYTGWMQ KWPTLQDLASASLEEVNQLWAGLGYYSRGRRLQEGARKVVEELGGHMPRTAETLQQLLPGVGRYTAGAIA SIAFGQATGVVDGNVARVLCRVRAIGADPSSTLVSQQLWGLAQQLVDPARPGDFNQAAMELGATVCTPQR PLCSQCPVESLCRARQRVEQEQLLASGSLSGSPDVEECAPNTGQCHLCLPPSEPWDQTLGVVNFPRKASR KPPREESSATCVLEQPGALGAQILLVQRPNSGLLAGLWEFPSVTWEPSEQLQRKALLQELQRWAGPLPAT HLRHLGEVVHTFSHIKLTYQVYGLALEGQTPVTTVPPGARWLTQEEFHTAAVSTAMKKVFRVYQGQQPGT CMGSKRSQVSSPCSRKKPRMGQQVLDNFFRSHISTDAHSLNSAAQ (MYH sequence 1) (SEQ ID NO: 366) MRKPRAAVGSGHRKQAASQEGRQKHAKNNSQAKPSACDAGLARQPEEVVLQASVSSYHLFRDVAEVTAFR GSLLSWYDQEKRDLPWRRRAEDEMDLDRRAYAVWVSEVMLQQTQVATVINYYTGWMQKWPTLQDLASASL EEVNQLWAGLGYYSRGRRLQEGARKVVEELGGHMPRTAETLQQLLPGVGRYTAGAIASIAFGQATGVVDG NVARVLCRVRAIGADPSSTLVSQQLWGLAQQLVDPARPGDFNQAAMELGATVCTPQRPLCSQCPVESLCR ARQRVEQEQLLASGSLSGSPDVEECAPNTGQCHLCLPPSEPWDQTLGVVNFPRKASRKPPREESSATCVL EQPGALGAQILLVQRPNSGLLAGLWEEPSVTWEPSEQLQRKALLQELQRWAGPLPATHLRHLGEVVHTFS HIKLTYQVYGLALEGQTPVTTVPPGARWLTQEEFHTAAVSTAMKKVFRVYQGQQPGTCMGSKRSQVSSPC SRKKPRMGQQVLDNFFRSHISTDAHSLNSAAQ (MYH sequence 2) (SEQ ID NO: 367) MRKPRAAVGSGHRKQAASQEGRQKHAKNNSQAKPSACDGLARQPEEVVLQASVSSYHLFRDVAEVTAFRG SLLSWYDQEKRDLPWRRRAEDEMDLDRRAYAVWVSEVMLQQTQVATVINYYTGWMQKWPTLQDLASASLE EVNQLWAGLGYYSRGRRLQEGARKVVEELGGHMPRTAETLQQLLPGVGRYTAGAIASIAFGQATGVVDGN VARVLCRVRAIGADPSSTLVSQQLWGLAQQLVDPARPGDFNQAAMELGATVCTPQRPLCSQCPVESLCRA RQRVEQEQLLASGSLSGSPDVEECAPNTGQCHLCLPPSEPWDQTLGVVNFPRKASRKPPREESSATCVLE QPGALGAQILLVQRPNSGLLAGLWEFPSVTWEPSEQLQRKALLQELQRWAGPLPATHLRHLGEVVHTFSH IKLTYQVYGLALEGQTPVTTVPPGARWLTQEEFHTAAVSTAMKKVFRVYQGQQPGTCMGSKRSQVSSPCS RKKPRMGQQVLDNFFRSHISTDAHSLNSAAQ (MYH sequence 3) (SEQ ID NO: 368) MTPLVSRLSRLWAIMRKPRAAVGSGHRKQAASQEGRQKHAKNNSQAKPSACDAGLARQPEEVVLQASVSS YHLFRDVAEVTAFRGSLLSWYDQEKRDLPWRRRAEDEMDLDRRAYAVWVSEVMLQQTQVATVINYYTGWM QKWPTLQDLASASLEEVNQLWAGLGYYSRGRRLQEGARKVVEELGGHMPRTAETLQQLLPGVGRYTAGAI ASIAFGQATGVVDGNVARVLCRVRAIGADPSSTLVSQQLWGLAQQLVDPARPGDFNQAAMELGATVCTPQ RPLCSQCPVESLCRARQRVEQEQLLASGSLSGSPDVEECAPNTGQCHLCLPPSEPWDQTLGVVNFPRKAS RKPPREESSATCVLEQPGALGAQILLVQRPNSGLLAGLWEFPSVTWEPSEQLQRKALLQELQRWAGPLPA THLRHLGEVVHTFSHIKLTYQVYGLALEGQTPVTTVPPGARWLTQEEFHTAAVSTAMKKVFRVYQGQQPG TCMGSKRSQVSSPCSRKKPRMGQQVLDNFFRSHISTDAHSLNSAAQ (MYH sequence 4) (SEQ ID NO: 369) PNKP See Table VI.7 APTX See Table VI.13

More specifically, in some embodiments, the down-regulator of the BER pathway may be an inhibitor of (e.g., an siRNA against) one or more factors of Table VI.17, or any combination thereof. Inhibitors of some of the proteins of Table VI.17 above are listed elsewhere in this specification.

VI.9 Nucleotide Excision Repair (NER)

The nucleotide excision repair (NER) pathway is an important excision mechanism that removes bulky helix-distorting lesions from DNA (see, e.g., Marteijn et al. (2014) NAT. REV. MOL. CELL BIOL. 15: 465-481). A brief description of NER is provided below.

NER a broad pathway encompassing two smaller pathways: global genomic NER (GG-NER) and transcription coupled repair NER (TC-NER). GG-NER and TC-NER use different factors for recognizing DNA damage. However, they utilize the same machinery for lesion incision, repair, and ligation.

Once damage is recognized, the cell removes a short single-stranded DNA segment that contains the lesion. Endonucleases XPF/ERCC1 and XPG (encoded by ERCC5) remove the lesion by cutting the damaged strand on either side of the lesion, resulting in a single-strand gap of 22-30 nucleotides. Next, the cell performs DNA gap filling synthesis and ligation. Involved in this process are: PCNA; RFC; DNA polymerase delta, DNA polymerase epsilon (Pol epsilon) and/or DNA Polymerase kappa (Pol kappa); and DNA ligase I and/or XRCC1/Ligase III. Replicating cells tend to use DNA polymerase epsilon and DNA ligase I, while non-replicating cells tend to use DNA polymerase delta, DNA polymerase kappa, and the XRCC1/Ligase III complex to perform the ligation step.

NER can involve one or more of the following factors: XPA-G, POLH, XPF, ERCC1, and LIG1. Transcription-coupled NER (TC-NER) can involve one or more of the following factors: CSA, CSB, XPB, XPD, XPG, ERCC1, and TTDA. Additional factors are shown in Table VI.18.

In some embodiments, the methods described herein involve down-regulating the NER pathway in order to promote HDR (e.g., HR, alt-HR or SSA). For instance, in some embodiments, the methods may involve modulating, e.g., inhibiting, a component (e.g., exactly one component, or one or more components, e.g., two or three components) of the NER pathway, e.g., a component of Table VI.18. More specifically, in some embodiments, the methods may involve modulating, e.g., inhibiting, exactly one of, or one or more of XPA-G, POLH, XPF, ERCC1, LIG1, CSA, CSB, XPA, XPB, XPC, XPD, XPF, XPG, ERCC1, TTDA, UVSSA, USP7, CETN2, RAD23B, UV-DDB, CAK subcomplex, RPA, and PCNA. Up-regulation of HDR, in some embodiments, is achieved using siRNA directed against one or more of the components listed in Table VI.1. In some embodiments, a NER pathway is down regulated using an HDR-enhancing gRNA that targets a component of a NER pathway, e.g., one or more of the components listed in Table VI.18 (e.g., one or more of XPA-G, POLH, XPF, ERCC1, LIG1, CSA, CSB, XPA, XPB, XPC, XPD, XPF, XPG, ERCC1, TTDA, UVSSA, USP7, CETN2, RAD23B, UV-DDB, CAK subcomplex, RPA, and PCNA). In some embodiments, the HDR-enhancing gRNA is used in combination with an eiCas9 molecule. In some embodiments, the HDR-enhancing gRNA is used in combination with an eaCas9 molecule. In some embodiments, the HDR-enhancing gRNA is administered to the cell as a HDR-enhancing gRNA:Cas9 molecule complex.

In some embodiments, one or more of HDR, alt-HR, anti-HR, NHEJ, MMEJ, SSA, SSBR, BER, and MMR are not substantially down-regulated, e.g., in some embodiments the only DNA damage repair pathway to be substantially down-regulated is the NER pathway.

TABLE VI.18 Factors Involved in NER. Factor Sequence XPA-G — POLH >sp|Q9Y253|POLH_HUMAN DNA polymerase eta OS = Homo sapiens GN = POLH PE = 1 SV = 1 (SEQ ID NO: 370) MATGQDRVVALVDMDCFFVQVEQRQNPHLRNKPCAVVQYKSWKGGGIIAVSYEARAFGVT RSMWADDAKKLCPDLLLAQVRESRGKANLTKYREASVEVMEIMSRFAVIERASIDEAYVD LTSAVQERLQKLQGQPISADLLPSTYIEGLPQGPTTAEETVQKEGMRKQGLFQWLDSLQI DNLTSPDLQLTVGAVIVEEMRAAIERETGFQCSAGISHNKVLAKLACGLNKPNRQTLVSH GSVPQLFSQMPIRKIRSLGGKLGASVIEILGIEYMGELTQFTESQLQSHFGEKNGSWLYA MCRGIEHDPVKPRQLPKTIGCSKNFPGKTALATREQVQWWLLQLAQELEERLTKDRNDND RVATQLVVSIRVQGDKRLSSLRRCCALTRYDAHKMSHDAFTVIKNCNTSGIQTEWSPPLT MLFLCATKFSASAPSSSTDITSFLSSDPSSLPKVPVTSSEAKTQGSGPAVTATKKATTSL ESFFQKAAERQKVKEASLSSLTAPTQAPMSNSPSKPSLPFQTSQSTGTEPFFKQKSLLLK QKQLNNSSVSSPQQNPWSNCKALPNSLPTEYPGCVPVCEGVSKLEESSKATPAEMDLAHN SQSMHASSASKSVLEVTQKATPNPSLLAAEDQVPCEKCGSLVPVWDMPEHMDYHFALELQ KSFLQPHSSNPQVVSAVSHQGKRNPKSPLACTNKRPRPEGMQTLESFFKPLTH >sp|Q9Y253-2|POLH_HUMAN Isoform 2 of DNA polymerase eta OS = Homo sapiens GN = POLH (SEQ ID NO: 371) MATGQDRVVALVDMDCFFVQVEQRQNPHLRNKPCAVVQYKSWKGGGIIAVSYEARAFGVT RSMWADDAKKLCPDLLLAQVRESRGKANLTKYREASVEVMEIMSRFAVIERASIDEAYVD LTSAVQERLQKLQGQPISADLLPSTYIEGLPQGPTTAEETVQKEGMRKQGLFQWLDSLQI DNLTSPDLQLTVGAVIVEEMRAAIERETGFQCSAGISHNKVLAKLACGLNKPNRQTLVSH GSVPQLFSQMPIRKIRSLGGKLGASVIEILGIEYMGELTQFTESQLQSHFGEKNGSWLYA MCRGIEHDPVKPRQLPKTIGCSKNFPGKTALATREQVQWWLLQLAQELEERLTKDRNDND RVATQLVVSIRVQGDKRLSSLRRCCALTRYDAHKMSHDAFTVIKNCNTSGIQTE XPF See Table VI.9 ERCC1 See Table VI.9 LIG1 See Table VI.9 CSA (also called >sp|Q13216|ERCC8_HUMAN DNA excision repair protein ERCC-8 OS = Homo ERCC8) sapiens GN = ERCC8 PE = 1 SV = 1 (SEQ ID NO: 372) MLGFLSARQTGLEDPLRLRRAESTRRVLGLELNKDRDVERIHGGGINTLDIEPVEGRYML SGGSDGVIVLYDLENSSRQSYYTCKAVCSIGRDHPDVHRYSVETVQWYPHDTGMFTSSSF DKTLKVWDTNTLQTADVFNFEETVYSHHMSPVSTKHCLVAVGTRGPKVQLCDLKSGSCSH ILQGHRQEILAVSWSPRYDYILATASADSRVKLWDVRRASGCLITLDQHNGKKSQAVESA NTAHNGKVNGLCFTSDGLHLLTVGTDNRMRLWNSSNGENTLVNYGKVCNNSKKGLKFTVS CGCSSEFVFVPYGSTIAVYTVYSGEQITMLKGHYKTVDCCVFQSNFQELYSGSRDCNILA WVPSLYEPVPDDDETTTKSQLNPAFEDAWSSSDEEG >sp|Q13216-2|ERCC8_HUMAN Isoform 2 of DNA excision repair protein ERCC-8 OS = Homo sapiens GN = ERCC8 (SEQ ID NO: 373) MLGFLSARQTGLEDPLRLRRAESTRRVLGLELNKDRDVERIHGGGINTLDIEPVEGRYML SGGSDGVIVLYDLENSSRQSYYTCKAVCSIGRDHPDVHRYSVETVQWYPHDTGMFTSSSF DKTLKVWDTNTLQTADVFNFEETVYSHHMSPVSTKHCLVAVGTRGPKVQLCDLKSGSCSH ILQGIFILFQTATTLSKRFNKKKRY CSB (also called >sp|Q03468|ERCC6_HUMAN DNA excision repair protein ERCC-6 OS = Homo ERCC6) sapiens GN = ERCC6 PE = 1 SV = 1 (SEQ ID NO: 374) MPNEGIPHSSQTQEQDCLQSQPVSNNEEMAIKQESGGDGEVEEYLSFRSVGDGLSTSAVG CASAAPRRGPALLHIDRHQIQAVEPSAQALELQGLGVDVYDQDVLEQGVLQQVDNAIHEA SRASQLVDVEKEYRSVLDDLTSCTTSLRQINKIIEQLSPQAATSRDINRKLDSVKRQKYN KEQQLKKITAKQKHLQAILGGAEVKIELDHASLEEDAEPGPSSLGSMLMPVQETAWEELI RTGQMTPFGTQIPQKQEKKPRKIMLNEASGFEKYLADQAKLSFERKKQGCNKRAARKAPA PVTPPAPVQNKNKPNKKARVLSKKEERLKKHIKKLQKRALQFQGKVGLPKARRPWESDMR PEAEGDSEGEESEYFPTEEEEEEEDDEVEGAEADLSGDGTDYELKPLPKGGKRQKKVPVQ EIDDDFFPSSGEEAEAASVGEGGGGGRKVGRYRDDGDEDYYKQRLRRWNKLRLQDKEKRL KLEDDSEESDAEFDEGFKVPGFLFKKLFKYQQTGVRWLWELHCQQAGGILGDEMGLGKTI QIIAFLAGLSYSKIRTRGSNYRFEGLGPTVIVCPTTVMHQWVKEFHTWWPPFRVAILHET GSYTHKKEKLIRDVAHCHGILITSYSYIRLMQDDISRYDWHYVILDEGHKIRNPNAAVTL ACKQFRTPHRIILSGSPMQNNLRELWSLFDFIFPGKLGTLPVFMEQFSVPITMGGYSNAS PVQVKTAYKCACVLRDTINPYLLRRMKSDVKMSLSLPDKNEQVLFCRLTDEQHKVYQNFV DSKEVYRILNGEMQIFSGLIALRKICNHPDLFSGGPKNLKGLPDDELEEDQFGYWKRSGK MIVVESLLKIWHKQGQRVLLFSQSRQMLDILEVFLRAQKYTYLKMDGTTTIASRQPLITR YNEDTSIFVFLLTTRVGGLGVNLTGANRVVIYDPDWNPSTDTQARERAWRIGQKKQVTVY RLLTAGTIEEKIYHRQIFKQFLTNRVLKDPKQRRFFKSNDLYELFTLTSPDASQSTETSA IFAGTGSDVQTPKCHLKRRIQPAFGADHDVPKRKKFPASNISVNDATSSEEKSEAKGAEV NAVTSNRSDPLKDDPHMSSNVTSNDRLGEETNAVSGPEELSVISGNGECSNSSGTGKTSM PSGDESIDEKLGLSYKRERPSQAQTEAFWENKQMENNFYKHKSKTKHHSVAEEETLEKHL RPKQKPKNSKHCRDAKFEGTRIPHLVKKRRYQKQDSENKSEAKEQSNDDYVLEKLFKKSV GVHSVMKHDAIMDGASPDYVLVEAEANRVAQDALKALRLSRQRCLGAVSGVPTWTGHRGI SGAPAGKKSRFGKKRNSNFSVQHPSSTSPTEKCQDGIMKKEGKDNVPEHFSGRAEDADSS SGPLASSSLLAKMRARNHLILPERLESESGHLQEASALLPTTEHDDLLVEMRNFIAFQAH TDGQASTREILQEFESKLSASQSCVFRELLRNLCTFHRTSGGEGIWKLKPEYC XPA >sp|P23025|XPA_HUMAN DNA repair protein complementing XP-A cells OS = Homo sapiens GN = XPA PE = 1 SV = 1 (SEQ ID NO: 375) MAAADGALPEAAALEQPAELPASVRASIERKRQRALMLRQARLAARPYSATAAAATGGMA NVKAAPKIIDTGGGFILEEEEEEEQKIGKVVHQPGPVMEFDYVICEECGKEFMDSYLMNH FDLPTCDNCRDADDKHKLITKTEAKQEYLLKDCDLEKREPPLKFIVKKNPHHSQWGDMKL YLKLQIVKRSLEVWGSQEALEEAKEVRQENREKMKQKKFDKKVKELRRAVRSSVWKRETI VHQHEYGPEENLEDDMYRKTCTMCGHELTYEKM XPB MGKRDRADRDKKKSRKRHYEDEEDDEEDAPGNDPQEAVPSAAGKQVDESGTKVDEYGAKD YRLQMPLKDDHTSRPLWVAPDGHIFLEAFSPVYKYAQDFLVAIAEPVCRPTHVHEYKLTA YSLYAAVSVGLQTSDITEYLRKLSKTGVPDGIMQFIKLCTVSYGKVKLVLKHNRYFVESC HPDVIQHLLQDPVIRECRLRNSEGEATELITETFTSKSAISKTAESSGGPSTSRVTDPQG KSDIPMDLFDFYEQMDKDEEEEEETQTVSFEVKQEMIEELQKRCIHLEYPLLAEYDFRND SVNPDINIDLKPTAVLRPYQEKSLRKMFGNGRARSGVIVLPCGAGKSLVGVTAACTVRKR CLVLGNSAVSVEQWKAQFKMWSTIDDSQICRFTSDAKDKPIGCSVAISTYSMLGHTTKRS WEAERVMEWLKTQEWGLMILDEVHTIPAKMFRRVLTIVQAHCKLGLTATLVREDDKIVDL NFLIGPKLYEANWMELQNNGYIAKVQCAEVWCPMSPEFYREYVAIKTKKRILLYTMNPNK FRACQFLIKFHERRNDKIIVFADNVFALKEYAIRLNKPYIYGPTSQGERMQILQNFKHNP KINTIFISKVGDTSFDLPEANVLIQISSHGGSRRQEAQRLGRVLRAKKGMVAEEYNAFFY SLVSQDTQEMAYSTKRQRFLVDQGYSFKVITKLAGMEEEDLAFSTKEEQQQLLQKVLAAT DLDAEEEVVAGEFGSRSSQASRRFGTMSSMSGADDTVYMEYHSSRSKAPSKHVHPLFKRF RK (XPB CCDS 2144.1) (SEQ ID NO: 376) XPC >sp|Q01831|XPC_HUMAN DNA repair protein complementing XP-C cells OS = Homo sapiens GN = XPC PE = 1 SV = 4 (SEQ ID NO: 377) MARKRAAGGEPRGRELRSQKSKAKSKARREEEEEDAFEDEKPPKKSLLSKVSQGKRKRGC SHPGGSADGPAKKKVAKVTVKSENLKVIKDEALSDGDDLRDFPSDLKKAHHLKRGATMNE DSNEEEEESENDWEEVEELSEPVLGDVRESTAFSRSLLPVKPVEIEIETPEQAKTRERSE KIKLEFETYLRRAMKRFNKGVHEDTHKVHLLCLLANGFYRNNICSQPDLHAIGLSIIPAR FTRVLPRDVDTYYLSNLVKWFIGTFTVNAELSASEQDNLQTTLERRFAIYSARDDEELVH IFLLILRALQLLTRLVLSLQPIPLKSATAKGKKPSKERLTADPGGSSETSSQVLENHTKP KTSKGTKQEETFAKGTCRPSAKGKRNKGGRKKRSKPSSSEEDEGPGDKQEKATQRRPHGR ERRVASRVSYKEESGSDEAGSGSDFELSSGEASDPSDEDSEPGPPKQRKAPAPQRTKAGS KSASRTHRGSHRKDPSLPAASSSSSSSKRGKKMCSDGEKAEKRSIAGIDQWLEVFCEQEE KWVCVDCVHGVVGQPLTCYKYATKPMTYVVGIDSDGWVRDVTQRYDPVWMTVTRKCRVDA EWWAETLRPYQSPFMDREKKEDLEFQAKHMDQPLPTAIGLYKNHPLYALKRHLLKYEATY PETAAILGYCRGEAVYSRDCVHTLHSRDTWLKKARVVRLGEVPYKMVKGFSNRARKARLA EPQLREENDLGLFGYWQTEEYQPPVAVDGKVPRNEFGNVYLFLPSMMPIGCVQLNLPNLH RVARKLDIDCVQAITGFDFHGGYSHPVTDGYIVCEEFKDVLLTAWENEQAVIERKEKEKK EKRALGNWKLLAKGLLIRERLKRRYGPKSEAAAPHTDAGGGLSSDEEEGTSSQAEAARIL AASWPQNREDEEKQKLKGGPKKTKREKKAAASHLFPFEQL >sp|Q01831-2|XPC_HUMAN Isoform 2 of DNA repair protein complementing XP-C cells OS = Homo sapiens GN = XPC (SEQ ID NO: 378) MARKRAAGGEPRGRELRSQKSKAKSKARREEEEEDAFEDEKPPKKSLLSKVSQGKRKRGC SHPGGSADGPAKKKVAKVTVKSENLKVIKDEALSDGDDLRDFPSDLKKAHHLKRGATMNE DSNEEEEESENDWEEAKTRERSEKIKLEFETYLRRAMKRFNKGVHEDTHKVHLLCLLANG FYRNNICSQPDLHAIGLSIIPARFTRVLPRDVDTYYLSNLVKWFIGTFTVNAELSASEQD NLQTTLERRFAIYSARDDEELVHIFLLILRALQLLTRLVLSLQPIPLKSATAKGKKPSKE RLTADPGGSSETSSQVLENHTKPKTSKGTKQEETFAKGTCRPSAKGKRNKGGRKKRSKPS SSEEDEGPGDKQEKATQRRPHGRERRVASRVSYKEESGSDEAGSGSDFELSSGEASDPSD EDSEPGPPKQRKAPAPQRTKAGSKSASRTHRGSHRKDPSLPAASSSSSSSKRGKKMCSDG EKAEKRSIAGIDQWLEVFCEQEEKWVCVDCVHGVVGQPLTCYKYATKPMTYVVGIDSDGW VRDVTQRYDPVWMTVIRKCRVDAEWWAETLRPYQSPFMDREKKEDLEFQAKHMDQPLPTA IGLYKNHPLYALKRHLLKYEATYPETAAILGYCRGEAVYSRDCVHTLHSRDTWLKKARVV RLGEVPYKMVKGFSNRARKARLAEPQLREENDLGLFGYWQTEEYQPPVAVDGKVPRNEFG NVYLFLPSMMPIGCVQLNLPNLHRVARKLDIDCVQAITGFDFHGGYSHPVTDGYIVCEEF KDVLLTAWENEQAVIERKEKEKKEKRALGNWKLLAKGLLIRERLKRRYGPKSEAAAPHTD AGGGLSSDEEEGTSSQAEAARILAASWPQNREDEEKQKLKGGPKKTKREKKAAASHLFPF EQL >sp|Q01831-3|XPC_HUMAN Isoform 3 of DNA repair protein complementing XP-C cells OS = Homo sapiens GN = XPC (SEQ ID NO: 379) MARKRAAGGEPRGRELRSQKSKAKSKARREEEEEDAFEDEKPPKKSLLSKVSQGKRKRGC SHPGGSADGPAKKKVAKVTVKSENLKVIKDEALSDGDDLRDFPSDLKKAHHLKRGATMNE DSNEEEEESENDWEEVEVKR XPD MKLNVDGLLVYFPYDYTYPEQFSYMRELKRTLDAKGHGVLEMPSGTGKTVSLLALIMAYQ RAYPLEVTKLIYCSRTVPEIEKVIEELRKLLNFYEKQEGEKLPFLGLALSSRKNLCIHPE VTPLRFGKDVDGKCHSLTASYVRAQYQHDTSLPHCRFYEEFDAHGREVPLPAGIYNLDDL KALGRRQGWCPYFLARYSILHANVVVYSYHYLLDPKIADLVSKELARKAVVVFDEAHNID NVCIDSMSVNLTRRTLDRCQGNLETLQKTVLRIKETDEQRLRDEYRRLVEGLREASAARE TDAHLANPVLPDEVLQEAVPGSIRTAEHFLGFLRRLLEYVKWRLRVQHVVQESPPAFLSG LAQRVCIQRKPLRFCAERLRSLLHTLEITDLADFSPLTLLANFATLVSTYAKGFTIIIEP FDDRTPTIANPILHFSCMDASLAIKPVFERFQSVIITSGTLSPLDIYPKILDFHPVTMAT FTMTLARVCLCPMIIGRGNDQVAISSKFETREDIAVIRNYGNLLLEMSAVVPDGIVAFFT SYQYMESTVASWYEQGILENIQRNKLLFIETQDGAETSVALEKYQEACENGRGAILLSVA RGKVSEGIDFVHHYGRAVIMFGVPYVYTQSRILKARLEYLRDQFQIRENDFLTFDAMRHA AQCVGRAIRGKTDYGLMVFADKRFARGDKRGKLPRWIQEHLTDANLNLTVDEGVQVAKYF LRQMAQPFHREDQLGLSLLSLEQLESEETLKRIEQIAQQL (XPD Isoform 1 CCDS 33049.1) (SEQ ID NO: 380) MRELKRTLDAKGHGVLEMPSGTGKTVSLLALIMAYQRAYPLEVTKLIYCSRTVPEIEKVI EELRKLLNFYEKQEGEKLPFLGLALSSRKNLCIHPEVTPLRFGKDVDGKCHSLTASYVRA QYQHDTSLPHCRFYEEFDAHGREVPLPAGIYNLDDLKALGRRQGWCPYFLARYSILHANV VVYSYHYLLDPKIADLVSKELARKAVVVFDEAHNIDNVCIDSMSVNLTRRTLDRCQGNLE TLQKTVLRIKETDEQRLRDEYRRLVEGLREASAARETDAHLANPVLPDEVLQEAVPGSIR TAEHFLGFLRRLLEYVKWRLRVQHVVQESPPAFLSGLAQRVCIQRKPLRFCAERLRSLLH TLEITDLADFSPLTLLANFATLVSTYAKGQAQHCGSSRNQKRSHP (XPD Isoform 2 CCDS 46112.1) (SEQ ID NO: 381) XPF See Table VI.9 XPG MGVQGLWKLLECSGRQVSPEALEGKILAVDISIWLNQALKGVRDRHGNSIENPHLLTLFH RLCKLLFFRIRPIFVFDGDAPLLKKQTLVKRRQRKDLASSDSRKTTEKLLKTFLKRQAIK TAFRSKRDEALPSLTQVRRENDLYVLPPLQEEEKHSSEEEDEKEWQERMNQKQALQEEFF HNPQAIDIESEDFSSLPPEVKHEILTDMKEFTKRRRTLFEAMPEESDDFSQYQLKGLLKK NYLNQHIEHVQKEMNQQHSGHIRRQYEDEGGFLKEVESRRVVSEDTSHYILIKGIQAKTV AEVDSESLPSSSKMHGMSFDVKSSPCEKLKTEKEPDATPPSPRILLAMQAALLGSSSEEE LESENRRQARGRNAPAAVDEGSISPRTLSAIKRALDDDEDVKVCAGDDVQTGGPGAEEMR INSSTENSDEGLKVRDGKGIPFTATLASSSVNSAEEHVASTNEGREPTDSVPKEQMSLVH VGTEAFPISDESMIKDRKDRLPLESAVVRHSDAPGLPNGRELTPASPTCTNSVSKNETHA EVLEQQNELCPYESKFDSSLLSSDDETKCKPNSASEVIGPVSLQETSSIVSVPSEAVDNV ENVVSFNAKEHENFLETIQEQQTTESAGQDLISIPKAVEPMEIDSEESESDGSFIEVQSV ISDEELQAEFPETSKPPSEQGEEELVGTREGEAPAESESLLRDNSERDDVDGEPQEAEKD AEDSLHEWQDINLEELETLESNLLAQQNSLKAQKQQQERIAATVTGQMFLESQELLRLFG IPYIQAPMEAEAQCAILDLTDQTSGTITDDSDIWLFGARHVYRNFFNKNKFVEYYQYVDF HNQLGLDRNKLINLAYLLGSDYTEGIPTVGCVTAMEILNEFPGHGLEPLLKFSEWWHEAQ KNPKIRPNPHDTKVKKKLRILQLTPGFPNPAVAEAYLKPVVDDSKGSFLWGKPDLDKIRE FCQRYFGWNRTKTDESLFPVLKQLDAQQTQLRIDSFFRLAQQEKEDAKRIKSQRLNRAVT CMLRKEKEAAASEIEAVSVAMEKEFELLDKAKGKTQKRGITNTLEESSSLKRKRLSDSKG KNTCGGFLGETCLSESSDGSSSEDAESSSLMNVQRRTAAKEPKTSASDSQNSVKEAPVKN GGATTSSSSDSDDDGGKEKMVLVTARSVFGKKRRKLRRARGRKRKT (XPG CCDS 32004.1) (SEQ ID NO: 382) ERCC1 See Table VI.9 TTDA >sp|Q6ZYL4|TF2H5_HUMAN General transcription factor IIH subunit 5 OS = Homo sapiens GN = GTF2H5 PE = 1 SV = 1 MVNVLKGVLIECDPAMKQFLLYLDESNALGKKFIIQDIDDTHVFVIAELVNVLQERVGEL MDQNAFSLTQK (SEQ ID NO: 383) UVSSA >sp|Q2YD98|UVSSA_HUMAN UV-stimulated scaffold protein A OS = Homo sapiens GN = UVSSA PE = 1 SV = 2 (SEQ ID NO: 384) MDQKLSKLVEELTTSGEPRLNPEKMKELKKICKSSEEQLSRAYRLLIAQLTQEHAEIRLS AFQIVEELFVRSHQFRMLVVSNFQEFLELTLGTDPAQPLPPPREAAQRLRQATTRAVEGW NEKFGEAYKKLALGYHFLRHNKKVDFQDTNARSLAERKREEEKQKHLDKIYQERASQAER EMQEMSGEIESCLTEVESCFRLLVPFDFDPNPETESLGMASGMSDALRSSCAGQVGPCRS GTPDPRDGEQPCCSRDLPASAGHPRAGGGAQPSQTATGDPSDEDEDSDLEEFVRSHGLGS HKYTLDVELCSEGLKVQENEDNLALIHAARDTLKLIRNKFLPAVCSWIQRFTRVGTHGGC LKRAIDLKAELELVLRKYKELDIEPEGGERRRTEALGDAEEDEDDEDFVEVPEKEGYEPH IPDHLRPEYGLEAAPEKDTVVRCLRTRTRMDEEVSDPTSAAAQLRQLRDHLPPPSSASPS RALPEPQEAQKLAAERARAPVVPYGVDLHYWGQELPTAGKIVKSDSQHRFWKPSEVEEEV VNADISEMLRSRHITFAGKFEPVQHWCRAPRPDGRLCERQDRLKCPFHGKIVPRDDEGRP LDPEDRAREQRRQLQKQERPEWQDPELMRDVEAATGQDLGSSRYSGKGRGKKRRYPSLTN LKAQADTARARIGRKVFAKAAVRRVVAAMNRMDQKKHEKFSNQFNYALN >sp|Q2YD98-2|UVSSA_HUMAN Isoform 2 of UV-stimulated scaffold protein A OS = Homo sapiens GN = UVSSA (SEQ ID NO: 385) MDEEVSDPISAAAQLRQLRDHLPPPSSASPSRALPEPQEAQKLAAERARAPVVPYGVDLH YWGQELPTAGKIVKSDSQHRFWKPSEVEEEVVNADISEMLRSRHITFAGKFEPVQHWCRA PRPDGRLCERQDRLKCPFHGKIVPRDDEGRPLDPEDRAREQRRQLQKQERPEWQDPELMR DVEAATGQDLGSSRYSGKGRGKKRRYPSLTNLKAQADTARARIGRKVFAKAAVRRVVAAM NRMDQKKHEKFSNQFNYALN USP7 >sp|Q93009|UBP7_HUMAN Ubiquitin carboxyl-terminal hydrolase 7 OS = Homo sapiens GN = USP7 PE = 1 SV = 2 (SEQ ID NO: 386) MNHQQQQQQQKAGEQQLSEPEDMEMEAGDTDDPPRITQNPVINGNVALSDGHNTAEEDME DDTSWRSEATFQFTVERFSRLSESVLSPPCFVRNLPWKIMVMPRFYPDRPHQKSVGFFLQ CNAESDSTSWSCHAQAVLKIINYRDDEKSFSRRISHLFFHKENDWCFSNFMAWSEVIDPE KGFIDDDKVTFEVFVQADAPHGVAWDSKKHTGYVGLKNQGATCYMNSLLQTLFFTNQLRK AVYMMPTEGDDSSKSVPLALQRVFYELQHSDKPVGIKKLIKSFGWEILDSFMQHDVQELC RVLLDNVENKMKGTCVEGTIPKLFRGKMVSYIQCKEVDYRSDRREDYYDIQLSIKGKKNI FESFVDYVAVEQLDGDNKYDAGEHGLQEAEKGVKFLTLPPVLHLQLMRFMYDPQTDQNIK INDRFEFPEQLPLDEFLQKTDPKDPANYILHAVLVHSGDNHGGHYVVYLNPKGDGKWCKF DDDVVSRCTKEEAIEHNYGGHDDDLSVRHCTNAYMLVYIRESKLSEVLQAVTDHDIPQQL VERLQEEKRIEAQKRKERQEAHLYMQVQIVAEDQFCGHQGNDMYDEEKVKYTVFKVLKNS SLAEFVQSLSQTMGFPQDQIRLWPMQARSNGTKRPAMLDNEADGNKTMIELSDNENPWTI FLETVDPELAASGATLPKFDKDHDVMLFLKMYDPKTRSLNYCGHIYTPISCKIRDLLPVM CDRAGFIQDTSLILYEEVKPNLTERIQDYDVSLDKALDELMDGDIIVFQKDDPENDNSEL PTAKEYFRDLYHRVDVIFCDKTIPNDPGFVVTLSNRMNYFQVAKTVAQRLNTDPMLLQFF KSQGYRDGPGNPLRHNYEGTLRDLLQFFKPRQPKKLYYQQLKMKITDFENRRSFKCIWLN SQFREEEITLYPDKHGCVRDLLEECKKAVELGEKASGKLRLLEIVSYKIIGVHQEDELLE CLSPATSRTFRIEEIPLDQVDIDKENEMLVTVAHFHKEVFGTFGIPFLLRIHQGEHFREV MKRIQSLLDIQEKEFEKFKFAIVMMGRHQYINEDEYEVNLKDFEPQPGNMSHPRPWLGLD HFNKAPKRSRYTYLEKAIKIHN >sp|Q93009-3|UBP7_HUMAN Isoform 3 of Ubiquitin carboxyl-terminal hydrolase 7 OS = Homo sapiens GN = USP7 (SEQ ID NO: 2237) MAGNHRLGLEAGDTDDPPRITQNPVINGNVALSDGHNTAEEDMEDDTSWRSEATFQFTVE RFSRLSESVLSPPCFVRNLPWKIMVMPRFYPDRPHQKSVGFFLQCNAESDSTSWSCHAQA VLKIINYRDDEKSFSRRISHLFFHKENDWGFSNFMAWSEVIDPEKGFIDDDKVIFEVFVQ ADAPHGVAWDSKKHIGYVGLKNQGATCYMNSLLQTLFFINQLRKAVYMMPTEGDDSSKSV PLALQRVFYELQHSDKPVGTKKLTKSFGWETLDSFMQHDVQELCRVLLDNVENKMKGTCV EGTIPKLFRGKMVSYIQCKEVDYRSDRREDYYDIQLSIKGKKNIFESFVDYVAVEQLDGD NKYDAGEHGLQEAEKGVKFLTLPPVLHLQLMRFMYDPQTDQNIKINDRFEFPEQLPLDEF LQKTDPKDPANYILHAVLVHSGDNHGGHYVVYLNPKGDGKWCKFDDDVVSRCTKEEAIEH NYGGHDDDLSVRHCTNAYMLVYIRESKLSEVLQAVTDHDIPQQLVERLQEEKRIEAQKRK ERQEAHLYMQVQIVAEDQFCGHQGNDMYDEEKVKYTVFKVLKNSSLAEFVQSLSQTMGFP QDQIRLWPMQARSNGTKRPAMLDNEADGNKTMIELSDNENPWTIFLETVDPELAASGAIL PKFDKDHDVMLFLKMYDPKTRSLNYCGHIYTPISCKIRDLLPVMCDRAGFIQDTSLILYE EVKPNLTERIQDYDVSLDKALDELMDGDIIVFQKDDPENDNSELPTAKEYFRDLYHRVDV IFCDKTIPNDPGFVVILSNRMNYFQVAKTVAQRLNIDPMLLQFFKSQGYRDGPGNPLRHN YEGTLRDLLQFFKPRQPKKLYYQQLKMKITDFENRRSFKCIWLNSQFREEEITLYPDKHG CVRDLLEECKKAVELGEKASGKLRLLEIVSYKIIGVHQEDELLECLSPATSRTFRIEEIP LDQVDIDKENEMLVTVAHFHKEVFGTFGIPFLLRIHQGEHFREVMKRIQSLLDIQEKEFE KFKFAIVMMGRHQYINEDEYEVNLKDFEPQPGNMSHPRPWLGLDHFNKAPKRSRYTYLEK AIKIHN CETN2 >sp|P41208|CETN2_HUMAN Centrin-2 OS = Homo sapiens GN = CETN2 PE = 1 SV = 1 (SEQ ID NO: 2238) MASNFKKANMASSSQRKRMSPKPELTEEQKQEIREAFDLFDADGTGTIDVKELKVAMRAL GFEPKKEEIKKMISEIDKEGTGKMNFGDFLTVMTQKMSEKDTKEEILKAFKLFDDDETGK ISFKNLKRVAKELGENLTDEELQEMIDEADRDGDGEVSEQEFLRIMKKTSLY RAD23B >sp|P54727|RD23B_HUMAN UV excision repair protein RAD23 homolog B OS = Homo sapiens GN = RAD23B PE = 1 SV = 1 (SEQ ID NO: 2239) MQVTLKTLQQQTFKIDIDPEETVKALKEKIESEKGKDAFPVAGQKLIYAGKILNDDTALK EYKIDEKNFVVVMVIKPKAVSTPAPATTQQSAPASTTAVISSITTIVAQAPTPVPALAPT STPASITPASATASSEPAPASAAKQEKPAEKPAETPVATSPTATDSTSGDSSRSNLFEDA TSALVTGQSYENMVTEIMSMGYEREQVIAALRASFNNPDRAVEYLLMGIPGDRESQAVVD PPQAASTGAPQSSAVAAAAATTTATTTTTSSGGHPLEFLRNQPQFQQMRQIIQQNPSLLP ALLQQIGRENPQLLQQISQHQEHFIQMLNEPVQEAGGQGGGGGGGSGGIAEAGSGHMNYI QVTPQEKEAIERLKALGFPEGLVIQAYFACEKNENLAANFLLQQNFDED >sp|P54727-2|RD23B_HUMAN Isoform 2 of UV excision repair protein RAD23 homolog B OS = Homo sapiens GN = RAD23B (SEQ ID NO: 2240) MVTKPKAVSTPAPATTQQSAPASTTAVTSSTTTTVAQAPTPVPALAPTSTPASITPASAT ASSEPAPASAAKQEKPAEKPAETPVATSPTATDSTSGDSSRSNLFEDATSALVTGQSYEN MVTEIMSMGYEREQVIAALRASFNNPDRAVEYLLMGIPGDRESQAVVDPPQAASTGAPQS SAVAAAAATTTATTTTTSSGGHPLEFLRNQPQFQQMRQIIQQNPSLLPALLQQIGRENPQ LLQQISQHQEHFIQMLNEPVQEAGGQGGGGGGGSGGIAEAGSGHMNYIQVTPQEKEATER LKALGFPEGLVIQAYFACEKNENLAANFLLQQNFDED UV-DDB >sp|Q92466|DDB2_HUMAN DNA damage-binding protein 2 OS = Homo sapiens GN = DDB2 PE = 1 SV = 1 (SEQ ID NO: 2241) MAPKKRPETQKTSEIVLRPRNKRSRSPLELEPEAKKLCAKGSGPSRRCDSDCLWVGLAGP QILPPCRSIVRTLHQHKLGRASWPSVQQGLQQSFLHTLDSYRILQKAAPFDRRATSLAWH PTHPSTVAVGSKGGDIMLWNFGIKDKPTFIKGIGAGGSITGLKFNPLNTNQFYASSMEGT TRLQDFKGNILRVFASSDTINIWFCSLDVSASSRMVVTGDNVGNVILLNMDGKELWNLRM HKKKVTHVALNPCCDWFLATASVDQTVKIWDLRQVRGKASFLYSLPHRHPVNAACFSPDG ARLLTTDQKSEIRVYSASQWDCPLGLIPHPHRHFQHLTPIKAAWHPRYNLIVVGRYPDPN FKSCTPYELRTIDVFDGNSGKMMCQLYDPESSGISSLNEFNPMGDTLASAMGYHILIWSQ EEARTRK >sp|Q92466-2|DDB2_HUMAN Isoform D1 of DNA damage-binding protein 2 OS = Homo sapiens GN = DDB2 (SEQ ID NO: 2242) MAPKKRPETQKTSEIVLRPRNKRSRSPLELEPEAKKLCAKGSGPSRRCDSDCLWVGLAGP QILPPCRSIVRTLHQHKLGRASWPSVQQGLQQSFLHTLDSYRILQKAAPFDRRATSLAWH PTHPSTVAVGSKGGDIMLWNFGIKDKPTFIKGAAWHPRYNLIVVGRYPDPNFKSCTPYEL RTIDVFDGNSGKMMCQLYDPESSGISSLNEFNPMGDTLASAMGYHILIWSQEEARTRK >sp|Q92466-3|DDB2_HUMAN Isoform D2 of DNA damage-binding protein 2 OS = Homo sapiens GN = DDB2 (SEQ ID NO: 2243) MAPKKRPETQKTSEIVLRPRNKRSRSPLELEPEAKKLCAKGSGPSRRCDSDCLWVGLAGP QILPPCRSIVRTLHQHKLGRASWPSVQQGLQQSFLHTLDSYRILQKAAPFDRRATSLAWH PTHPSTVAVGSKGGDIMLWNFGIKDKPTFIKGHLVL >sp|Q92466-4|DDB2_HUMAN Isoform D3 of DNA damage-binding protein 2 OS = Homo sapiens GN = DDB2 (SEQ ID NO: 2244) MAPKKRPETQKTSEIVLRPRNKRSRSPLELEPEAKKLCAKGSGPSRRCDSDCLWVGLAGP QILPPCRSIVRTLHQHKLGRASWPSVQQIGAGGSITGLKFNPLNTNQFYASSMEGTTRLQ DFKGNILRVFASSDTINIWFCSLDVSASSRMVVTGDNVGNVILLNMDGKELWNLRMHKKK VTHVALNPCCDWFLATASVDQTVKIWDLRQVRGKASFLYSLPHRHPVNAACFSPDGARLL TTDQKSEIRVYSASQWDCPLGLIPHPHRHFQHLTPIKAAWHPRYNLIVVGRYPDPNFKSC TPYELRTIDVFDGNSGKMMCQLYDPESSGISSLNEFNPMGDTLASAMGYHILIWSQEEAR TRK >sp|Q92466-5|DDB2_HUMAN Isoform D4 of DNA damage-binding protein 2 OS = Homo sapiens GN = DDB2 (SEQ ID NO: 2245) MAPKKRPETQKTSEIVLRPRNKRSRSPLELEPEAKKLCAKGSGPSRRCDSDCLWVGLAGP QILPPCRSIVRTLHQHKLGRASWPSVQQGLQQSFLHTLDSYRILQKAAPFDRRATSLAWH PTHPSTVAVGSKGGDIMLWNFGIKDKPTFIKGIGAGGSITGLKFNPLNTNQFYASSMEGT TRLQDFKGNILRVFASSDTINIWFCSLDVSASSRMVVTGDNVGNVILLNMDGKELVSVPM EPGS CAK subcomplex — RPA — PCNA VI.13

More specifically, in some embodiments, the down-regulator of the NER pathway may be an inhibitor of (e.g., an siRNA against) one or more factors of Table VI.18, or any combination thereof. Inhibitors of some of the proteins of Table VI.18 above are listed elsewhere in this specification.

VI.10 Other Pathways

Several other DNA repair pathways exist in mammals. A dedicated pathway called the ICL repair pathway repairs interstrand crosslinks. ICL repair can involve the following factors: XPF and RAD51C. Translesion synthesis (TLS) is a pathway for repairing a single strand break left after a defective replication event. Error-free postreplication repair (PRR) is another pathway for repairing a single strand break left after a defective replication event.

In some embodiments, the methods herein do not substantially inhibit one or more of, e.g., all of, ICL repair, TLS, and PRR.

VI.11 The Role of Epigtentic Modification in DNA Repair

Breaks, e.g., DSBs, are characterized by distinct chromatin structure. This chromatin structure affects DNA repair.

In some embodiments, HDR repair is promoted by modulating the chromatin structure at the site of the DNA lesion. For example, in some embodiments, a Cas9 molecule and gRNA can induce a DSB in a desired location during G2 or another phase of the cycle. This DSB can be formed using, e.g., one Cas9 molecule with the ability to produce DSBs, or two nickases. In S/G2, a chromatin structure modulator can increase the likelihood that a DSB is repaired by HDR. A template nucleic acid can be added to the cell, so that the HDR machinery repairs the DSB using the template nucleic acid.

Upon formation of a double strand break, ATM and MDC1 (which can activate canonical NHEJ and HDR) allow recruitment of gamma-H2AX, a specialized histone, to a 1-2 megabase region surrounding the break. When no damage is detected, gamma-H2AX is kept in an inactive, phosphorylated state by the kinase WSTF. Upon damage sensing, EYA1 and EYA2 dephosphorylate and activate gamma-H2AX. Once gamma-H2AX is recruited to the break, it promotes repair via a number of DNA repair factors.

For instance, gamma-H2AX helps to prolong the association of NBS1, BRCA1, and 53BP1 at DSB regions (after their initial recruitment by PARP1). This effect may be mediated through a host of chromatin modifying enzymes including INO80 (to remove nucleosomes) and SWI/SNF (to relax the chromatin). MCPH1 may assist gamma-H2AX in stabilizing SWI/SNF association near the damage site.

Another chromatin modification at DNA damage sites is ubiquitination of K63 of H2A and gamma-H2AX. Ubiquitination (mono- or poly-ubiquitination) of K63 is performed by the ubiquitin ligase RNF8, which in turn recruits the BRCA1-A complex. This complex also has ubiquitin ligase activity (due to the BRE subunit, the BRCC36 subunit, and NAB1).

Sumoylation is another important modification of proteins near the break site, because it promotes association of BRCA1-A complex. PIAS1, a SUMO ligase, catalyzes sumoylation of BRCA1 to stimulate its ubiquitin ligase activity. PIAS4, another SUMO ligase, catalyzes sumoylation of 53BP1.

Sometimes a combination of histone modifications is required to recruit a factor. For example, 53BP1 contains Tudor domains that bind to mono- and dimethylated H4K20 and acetylated H4K16, as well as a motif that binds to ubiquitinylated H2AK15. Although methylated H2K20 is abundant in the genome, in the absence of DNA damage it is masked by JMJD2A (also referred to as KDM4A). The masking proteins are removed from damaged regions, allowing the recruitment of 53BP1. As to the H4K16 acetylation, this seems to be provided by HDAC1 and HDAC2, making these proteins promoters of the canonical NHEJ pathway.

Sites of DNA damage or lesions become enriched in histone methyltransferases complexes (e.g., polycomb proteins, histone deacetylases such as HDACs and sirtuins, and DNA methyltransferases).

DNA repair in heterochromatin requires loosening the tightly packed chromatin. When DNA is damaged, CHK2 phosphorylates HP1, causing HP1 to disassociate it from its usual position on the methylated K9 of histone H3. Further relaxation of the chromatin occurs when TIP60 (KAT5) acetylates H3, H4, and gamma-H2AX. Tip60 is recruited to double strand breaks and inhibits 53BP1 association. KAP1 also localizes to damaged heterochromatin, where it promotes chromatin relaxation. Another repair factor that localizes to damaged heterochromatin is EXPAND1, a chromatin remodeling factor that deconsenses chromatin. EXPAND1 is recruited to DSBs in a 53BP1-dependent manner.

Accordingly, in some embodiments, the methods herein involve modifying chromatin in order to promote HDR (e.g., HR, SSA or alt-HR). For instance, the methods may involve modulating, e.g., inhibiting, a chromatin-modifying factor (e.g., exactly one factor, or one or more factors). More specifically, the methods may involve modulating, e.g., inhibiting, exactly one of, or one or more of gamma-H2AX, WSTF, EYA1, EYA2, INO80, SWI/SNF, MCPH1, a ubiquitin ligase, RNF8, BRCA1, BRCA1-A, a SUMO ligase, PIAS1, JMJD2A, a histone deacetylase, HDAC1, HDAC2, CHK2, TIP60, KAP1, EXPAND1, or a component of Table VI.1(I), or any combination thereof. In some embodiments, the methods involve promoting HDR by inhibiting exactly one of, or one or more of MDC1, HDAC1, HDAC2. Up-regulation of HDR, in some embodiments, is achieved using siRNA directed against one or more of gamma-H2AX, WSTF, EYA1, EYA2, INO80, SWI/SNF, MCPH1, a ubiquitin ligase, RNF8, BRCA1, BRCA1-A, a SUMO ligase, PIAS1, JMJD2A, a histone deacetylase, HDAC1, HDAC2, CHK2, TIP60, KAP1, EXPAND1, or a component of Table VI.1(I), or any combination thereof. In some embodiments, a chromatin-modifying factor is down regulated using an HDR-enhancing gRNA that targets a chromatin-modifying factor, e.g., one or more of the components listed in Table VI.18 (e.g., one or more of gamma-H2AX, WSTF, EYA1, EYA2, INO80, SWI/SNF, MCPH1, a ubiquitin ligase, RNF8, BRCA1, BRCA1-A, a SUMO ligase, PIAS1, JMJD2A, a histone deacetylase, HDAC1, HDAC2, CHK2, TIP60, KAP1, EXPAND1, or a component of Table VI.1(I), or any combination thereof). In some embodiments, the HDR-enhancing gRNA is used in combination with an eiCas9 molecule. In some embodiments, the HDR-enhancing gRNA is used in combination with an eaCas9 molecule. In some embodiments, the HDR-enhancing gRNA is administered to the cell as a HDR-enhancing gRNA:Cas9 molecule complex.

In some embodiments, modifying the chromatin results in substantial inhibition of only DNA damage repair pathway e.g., exactly one of HDR, alt-HR, anti-HR, NHEJ, alt-NHEJ, MMEJ, SSBR, or SSA.

More specifically, in some embodiments, the chromatin structure modifier is an EZH2 inhibitor e.g., EPZ-6438 or an HDAC inhibitor e.g., TCA, or any combination thereof. In some embodiments, the EZH2 inhibitor modifies, e.g., inhibits, histone H3 trimethylation of residue K27. In some embodiments, the HDAC inhibitor modifies, e.g., promotes, histone H4 acetylation at residue K16. In some embodiments, an HDAC inhibitor does not interfere with HDR, e.g., the HDAC inhibitor does not interfere with recruitment of factors that promote HDR.

TABLE VII HDR-enhancers that are chromatin structure modifiers 1. EZH2 Compounds Phase Structure GSK343

EPZ-6438 Phase I/II

GSK2816126 Phase I SureCN6120847

EPZ005687

siRNAs Accell Human EZH2 siRNA, sold by Dharmacon; EZH2 Silencer, sold by Life Technologies antibodies EZH2 monoclonal antibody (M07), clone 1D11, sold by Abnova Ezh2 Antibody, sold by Cell Signaling Technology 2. HDAC-HDACI & II inhibitors Compounds Trichostatin A (TSA) Sodium Butyrate (NaB) siRNA HDAC1 CAGCGACUGUUUGAGAACC (sense) (SEQ ID NO: 2246) CUAAUGAGCUUCCAUACAA (sense) (SEQ ID NO: 2247) HDAC2 Accell Human HDAC2 siRNA, sold by Dharmacon; HDAC2 Silencer, sold by Life Technologies; GCGGAUAGCUUGUGAUGAA (sense); (SEQ ID NO: 2248) GCAAAGAAAGCUAGAAUUG (sense) (SEQ ID NO: 2249) antibodies HDAC2 HDAC2 Antibody, sold by Cell Signalling Technology HDAC2 Antibody, sold by Novus Biologicals 3. Histone Demethylation KDM4A/JMJD2A siRNA Commercially available from Dharmacon or Ambion 3. Histone Methylation Setd2 siRNA Commercially available from Dharmacon or Ambion

In some embodiments, one or more of HDR, alt-HR, anti-HR, NHEJ, MMEJ, SSA, SSBR, MMR, NER, and BER are not substantially down-regulated. In some embodiments, only one DNA damage repair pathway is down-regulated.

VI.12 Additional Considerations in Selecting an HDR-Enhancer

In some embodiments, even though a component is typically recognized as participating in two pathways, it is possible to inhibit that component, resulting in inhibition of only one of the pathways. For example, in some embodiments, a component promotes a first pathway and inhibits a second pathway. In this embodiment, an inhibitor of the component would inhibit the first pathway but not the second. As a second example, in some embodiments, if a component is essential to a first pathway but redundant to a second pathway, inhibiting the component would inhibit the first pathway but not the second. As a third example, in some embodiments, a component may have a first activity that promotes a first pathway and a second activity that promotes a second pathway. In this embodiment, an inhibitor of the component's first activity would inhibit the first pathway but not the second. As a fourth example, in some embodiments, a component might act in a first pathway and a second pathway, and a low dose of an inhibitor inhibits the first pathway, and a higher dose of the inhibitor is necessary to inhibit the second pathway.

In some cases, the HDR-enhancer molecule is an antibody. Several commercially available antibodies for use in the methods disclosed herein are known in the art. It is understood that when using an antibody, and especially when administering the antibody to a human patient, one can use an antibody designed to minimize the patient's immune response against the antibody. For instance, a human antibody or a humanized antibody can be used. Methods of designing humanized antibodies are known in the art. It is also understood that one can use an antibody, or an antigen binding fragment thereof, that comprises CDRs (e.g., one or more of, e.g., all of CDRs HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, using the Kabat or Chothia definitions of CDRs) of an antibody described herein. The antibody can be, e.g., polyclonal or monoclonal, or an antigen binding portion thereof. The antibody can be, e.g., an IgG, IgM, IgA, IgD, or IgE molecule or an antigen-binding portion thereof. The antigen-binding portion may comprise a Fab, a F(ab′)2, Fv, disulfide-linked F, scFv, single domain antibody, closed conformation multispecific antibody, disulfide-linked dscFV, or diabody. The antibody may be created by any suitable system; for example, it may be isolated from the serum of an animal, produced in a cell e.g., a hybridoma, a yeast cell, or a bacterial cell.

In some embodiments, the antibody is an intrabody. An intrabody is an antibody that can bind to an intracellular protein. To achieve intracellular localization, the introbody may comprise one or more localization moieties, e.g., a nuclear localization signal (NLS). In some embodiments the intrabody is produced in the cell that is its intended destination, e.g., the cell comprising the target nucleic acid to be edited. In other embodiments, the intrabody is produced in a host cell, e.g., a cultured cell, e.g., a mammalian, eukaryotic, or bacterial cell. In some embodiments, the intrabody comprises an scFv.

In some embodiments, the HDR-enhancer molecule is an siRNA. It is understood that when using a siRNA, and especially when administering the siRNA to a human patient, one can use a siRNA designed to promote the stability of the siRNA, for example by using chemically modified siRNA. Numerous modifications are known and are discussed in more detail in Section X herein. For instance, the siRNA may comprise one or more modified bases, modifications to the backbone, and/or moieties conjugated to the nucleic acid.

In some embodiments, the HDR-enhancer molecule is an HDR-enhancing gRNA. In some embodiments, the HDR-enhancing gRNA is used in combination with an eiCas9 molecule. In some embodiments, when the HDR-enhancing gRNA is used in combination with an eiCas9, the eiCas9 molecule will temporarily or transiently prevent and/or reduce transcription of the targeted gene in the cell, as compared to the level of transcription of the targeted gene in a cell that does not comprise the HDR-enhancing gRNA and eiCas9 molecule. Temporary inhibition of the targeted gene will allow the other Cas9 system to target the gene of interest and promote the repair of the target DNA via an HDR pathway, e.g., anti-HR, HR, and/or SSA. In some embodiments, the HDR-enhancing gRNA is used in combination with an eaCas9 molecule. In some embodiments, the HDR-enhancing gRNA is administered to the cell as a HDR-enhancing gRNA:Cas9 molecule complex.

In some embodiments, the HDR-enhancer molecule is an anti-MiR. An anti-MiR is an antisense nucleic acid that is capable of inhibiting a microRNA. In some embodiments, an anti-MiR is 100% complementary to the microRNA it targets, or a portion thereof. The anti-MiR may comprise RNA, and may comprise chemical modifications.

In some embodiments, HDR-enhancer has at least 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 97, 98, 99 or 100% homology with, or differs by no more than 50, 40, 30, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, amino acid residues from a naturally occurring protein described herein, e.g., a protein of any of Tables VI.1, VI.2, VI.4, VI.7, VI.9, VI.11, VI.13, VI.15, VI.17, and VI.18.

In some embodiments, the DNA damage response (DDR) pathway is not modulated. For example, in some embodiments, levels and activity of ATM, ATR, CHK1, and CHK2 are not altered. While not wishing to be bound by theory, in some embodiments leaving these components unaltered avoids certain side-effects.

In some embodiments, the HDR-enhancer modulates, e.g., inhibits, one or more of: ABL1, ATM, ATR, AURKB, BACH1, BARD1, BCCIP, BLM, BRCA2, BRCC3, BRE, BUB1B, C11orf30, CCNA2, CDC45, CDK1, CDK2, CDK4, CHEK1, CHEK2, DMC1, ECD, FANCD2, FANCE, FANCG, FANCI, FLNA, FYN, GRB2, H2AFX, HDAC1, HDAC2, HMG20B, KAT2B, KIF4A, LMNA, MCPH1, MGMT, MLH1, MLH3, MND1, MORF4L1, MRE11A, MSH4, MTA2, PALB2, PCNA, PDS5B, PLK1, PMS1, PMS2, PSMC3IP, PSMD3, PSMD6, RAD21, RAD23A, RAD50, RAD51, RAD51B, RAD51C, RBBP8, RPA1, RPA2, RPA3, SERPINH1, SHFM1, SIRT1, SIRT2, SKP2, SMAD1, SMAD2, SMAD3, SMC3, SP1, SPO11, STAT5A, SYCP3, TEX15, TOP3A, TP53, UBC, UQCC1, USP11, WDR16, and XRCC3.

In some embodiments, the HDR-enhancer does not modulate, e.g., does not inhibit, one or more of: ABL1, ATM, ATR, AURKB, BACH1, BARD1, BCCIP, BLM, BRCA2, BRCC3, BRE, BUB1B, C11orf30, CCNA2, CDC45, CDK1, CDK2, CDK4, CHEK1, CHEK2, DMC1, ECD, FANCD2, FANCE, FANCG, FANCI, FLNA, FYN, GRB2, H2AFX, HDAC1, HDAC2, HMG20B, KAT2B, KIF4A, LMNA, MCPH1, MGMT, MLH1, MLH3, MND1, MORF4L1, MRE11A, MSH4, MTA2, PALB2, PCNA, PDS5B, PLK1, PMS1, PMS2, PSMC3IP, PSMD3, PSMD6, RAD21, RAD23A, RAD50, RAD51, RAD51B, RAD51C, RBBP8, RPA1, RPA2, RPA3, SERPINHI, SHFM1, SIRT1, SIRT2, SKP2, SMAD1, SMAD2, SMAD3, SMC3, SP1, SPO11, STAT5A, SYCP3, TEX15, TOP3A, TP53, UBC, UQCC1, USP11, WDR16, and XRCC3.

VI.13 Promoting HDR by Modulating the Cell Cycle

Since the HDR machinery is active during G2, in some aspects, the present disclosure provides methods of promoting HDR by increasing the proportion of cells in G2, e.g., by administering an agent that arrests cells in G2. In some embodiments, this agent induces reversible cell cycle arrest, so that cells can divide normally once the inhibitor is no longer effective. The agent may be, e.g., an inhibitor of a cyclin-dependent kinase, e.g., an inhibitor of CDK1. Inhibition of Cdk1 can arrest reversibly cells at the G2/M boundary. Exemplary cell cycle arrest agents are provided in Table VIII below.

TABLE VI.20 Cell cycle arrest agents. 1. Cdk1 Compounds Reference(s) RO-3306 Vassilev et al. (2006) PROC. NAT'L. ACAD. SCI. USA 103(28): 10660-5. AZD 5438 Camidge et al. (2006) Cancer Chemother. Pharmacol. 60(4): 479-88.

In some embodiments, a cell is treated with a cell cycle arrest agent in combination with another HDR-enhancer, e.g., an HDR-enhancer as described in one or more of Sections VI.1 through VI.13 above.

VII. Creating Mutations, e.g., Deletions, by Promoting Error-Prone DNA Repair

The present disclosure provides, e.g., compositions and methods for creating mutations, e.g., deletions, in a target region. In some embodiments, a mutation, e.g., deletion is created by making a Cas9-mediated break at the location where the deletion is desired, and inhibiting a DNA damage repair pathway so that the break is repaired by an error-prone pathway such as alt-NHEJ. In the wild-type context, alt-NHEJ occurs when there is a small degree of resection at the break. (When there is no resection, canonical NHEJ is favored, and when there is a large degree of resection, HDR or SSA is favored.) Accordingly, in the methods described in this section, it is often desirable to induce a short amount of resection.

Generally, in order to promote alt-NHEJ, one does not provide a template nucleic acid, because a template nucleic acid causes a cell to favor HDR over alt-NHEJ.

In some embodiments, the error-prone pathway is promoted by down-regulating HDR. Down-regulators of HDR are listed herein, e.g., in Section VI.1, e.g., in Table VI.3. In some embodiments, the methods herein involve down-regulating an HDR pathway, in the absence of a template nucleic acid, in order to promote alt-NHEJ. For instance, the methods may involve modulating, e.g., inhibiting, a component (e.g., exactly one component, or one or more components, e.g., two or three components) of an HDR pathway, e.g., a component of Table VI.1(C) or VI.2. In embodiments, HDR is down-regulated using an agent of Table VI.3. In embodiments where HDR is inhibited, this disclosure contemplates inhibiting downstream components of HDR, e.g., BRCA2 and/or RAD51. In some embodiments, BRCA1 is inhibited, in order to result in a loss of resection, to up-regulate the NHEJ pathway (see, e.g., Cotta-Ramusino et al. (2011) SCIENCE 332(6035): 1313-7). In some embodiments, upstream components of HDR, e.g., one or more pro-resection proteins, e.g., exonucleases, are not inhibited.

In some embodiments, resection is promoted by inhibiting an anti-resection protein. Down-regulators of anti-resection proteins are listed, e.g., in Section VI.4, e.g., in Table VI.8. Accordingly, in some embodiments, a cell is treated with a (e.g., exactly one, or one or more) inhibitor of an anti-resection protein.

In embodiments, resection is promoted by providing a protein that promotes resection, e.g., an exonuclease. In some embodiments, the method comprises providing (e.g., by overexpressing) MRE11, NBS1, BRCA1-C, CtIP, MRN, EXO1, BLM, or DNA2 endonuclease.

The method of creating a deletion may be used during any time that the alt-NHEJ machinery is active, e.g., in G1, S, or G2 phase.

In some embodiments, a mutation, e.g., a deletion, is created by making a Cas9-mediated break at the location where the deletion is desired, and inhibiting a DNA damage repair pathway so that the break is repaired by an error-prone pathway, e.g., C-NHEJ. While not wishing to be bound by theory, in some embodiments, inhibition of BRCA2 and Rad51 decreases other HDR pathways without perturbing resection, and therefore the SSA repair pathway becomes more dominant. (see, e.g., Cotta-Ramusino et al. (2011); and Stark et al. (2004) MOL. CELL BIOL. 24(21): 9305-16).

In some embodiments, the error prone pathway that is promoted is SSA. In some embodiments, a template nucleic acid is not provided to resolve a DNA lesion because a template nucleic acid causes a cell to favor other HDR pathways over SSA. Thus, in some embodiments, one HDR pathway is perturbed, and no template nucleic acid is provided, in order promote SSA. In addition, in some embodiments, a cell is treated with an up-regulator (e.g., exactly one up-regulator, or one or more up-regulators) of SSA. In embodiments, EPR-enhancer has at least 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homology with, or differs by no more than 50, 40, 30, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, amino acid residues from a naturally occurring protein described herein, e.g., a protein of any of Table VI.11 which lists components of the SSA pathway.

In some embodiments, a cell is treated with a down-regulator (e.g., exactly one down-regulator, or one or more down-regulators) of HDR.

In some embodiments, the SDMMJ pathway is down-regulated in order to promote SSA. For instance, DNA polymerase theta (Pol Theta) can be inhibited in order to down-regulate SDMMJ. Experiments that abrogate Pol Theta expression result in larger deletions, a phenotype that resembles SSA. Accordingly, in some embodiments, a cell is treated with a down-regulator (e.g., exactly one down-regulator, or one or more down-regulators) of SDMMJ.

In one embodiment, the EPR enhancer is an siRNA. In another embodiment, the EPR enhancer is an antibody, e.g., an intrabody. In another embodiment, the EPR enhancer is an EPR-enhancing gRNA. As used herein, the term “EPR-enhancing gRNA” refers to a gRNA, which, in combination with a Cas9 molecule (e.g., an eiCas9 molecule), enhances (e.g., increases the frequency or efficiency of) error-prone repair (e.g., alt-NJEH and SSA). In some embodiments, the EPR-enhancing gRNA guides a Cas9-mediated reduction in the transcription of a gene encoding a non-error-prone DNA damage repair pathway protein. In some embodiments, the EPR-enhancing gRNA guides a Cas9-mediated cleavage event in a gene encoding a non-error-prone DNA damage repair pathway protein (e.g., a protein involved in HDR, such as HR, alt-HR, and/or SSA).

The methods in this section may be used in a variety of contexts. For example, they may be used to, e.g., inactivate a preselected gene in a model organism in order to study the gene. In addition, the methods may be used to inactivate a gene that causes disease, e.g., an oncogene or a gene of a pathogen (e.g., a viral gene that has integrated into a host cell's genome, a protease, a polymerase, a structural gene, a bacterial gene, a toxin, a cell wall synthesis gene, or a drug resistance gene).

VIII. Target Cells

Cas9 molecules and gRNA molecules, e.g., a Cas9 molecule/gRNA molecule complex, as disclosed herein, can be used to manipulate e.g., to edit a target nucleic acid, a wide variety of cells. Additional details on types of cells that can be manipulated may be found in the section entitled “VIIA. TARGETS: CELLS” of PCT Application WO2015/048577, the entire contents of which are expressly incorporated herein by reference.

In some embodiments, a cell, or a population of cells, is manipulated by editing (e.g., introducing a mutation or correcting) one or more target genes, e.g., as described herein. In an embodiment, a cell, or a population of cells, is manipulated by editing one or more non-coding sequences, e.g., an alteration in an intron or in a 5′ or 3′ non-translated or non-transcribed region. In an embodiment, a cell is manipulated by editing the sequence of a control element, e.g., a promoter, enhancer, or a cis-acting or trans-acting control element. In an embodiment, a cell, or a population of cells, is manipulated by editing one or more coding sequences, e.g., an alteration in an exon. In some embodiments, a cell, or a population of cells, is manipulated in vitro. In other embodiments, a cell, or a population of cells, is manipulated ex vivo. In some embodiments, a cell, or a population of cells, is manipulated in vivo. In some embodiments, the expression of one or more target genes (e.g., one or more target genes described herein) is modulated, e.g., in vivo. In other embodiments, the expression of one or more target genes (e.g., one or more target genes described herein) is modulated, e.g., ex vivo. In other embodiments, the expression of one or more target genes (e.g., one or more target genes described herein) is modulated, e.g., in vitro.

In some embodiments, the cell, or population of cells, is a T cell, e.g., a CD8⁺ T cell (e.g., a CD8⁺ naïve T cell, central memory T cell, or effector memory T cell), a CD4⁺ T cell, a stem cell memory T cell, a helper T cell, a regulatory T cell, a cytotoxic T cell, a natural killer T cell, or a population of such cells.

In some embodiments, the cell, or population of cells, is a Hematopoietic Stem Cell (e.g., a long term hematopoietic stem cell (LT-HSC), a short term hematopoietic stem cell (ST-HSC), a multipotent progenitor (MPP) cell, a lineage restricted progenitor (LRP) cell (e.g., a lymphoid progenitor cell, a myeloid progenitor cell (e.g., a common myeloid progenitor (CMP) cell), an erythroid progenitor cell (e.g., a megakaryocyte erythroid progenitor (MEP) cell)), or a population of such cells.

In some embodiments, the cell, or population of cells, is a retinal cell (e.g., a photoreceptor cell (e.g., a rod cell, a cone cell) a retinal pigmented epithelium (RPE) cell), a trabecular meshwork cell, or a population of such cells.

In some embodiments, the cell, or population of cells, is a cochlear hair cell (e.g., an outer hair cell or an inner hair cell), or a population of cochlear hair cells.

In some embodiments, the cell, or population of cells, is a pulmonary epithelial cell (e.g., a bronchial epithelial cell or an alveolar epithelial cell), a pulmonary epithelial progenitor cell, or a population of such cells.

In some embodiments, the cell, or population of cells, is a striated muscle cell, a cardiac muscle cell, a muscle satellite cell, or a population of such cells.

In some embodiments, the cell, or population of cells, is a neuron, a neuronal stem cell, or a population of such cells.

In some embodiments, the cell, or population of cells, is a mesenchymal stem cell, or a population of mesenchymal stem cells.

In some embodiments, the cell, or population of cells, is an induced pluripotent stem (iPS) cell, or a population of iPs cells.

In some embodiments, the cell, or population of cells, is an embryonic stem cell, or a population of embryonic stem cells.

In some embodiments, the cell, or population of cells, is a monocyte, a megakaryocyte, a neutrophil, an eosinophil, a basophil, a mast cell, a reticulocyte, or a population of such cells.

In some embodiments, the cell, or population of cells, is a B cell, e.g., a progenitor B cell, a Pre B cell, a Pro B cell, a memory B cell, or a plasma B cell, or a population of B cells.

In some embodiments, the cell, or population of cells, is a gastrointestinal epithelial cell, a biliary epithelial cell, a pancreatic ductal epithelial cell, an intestinal stem cell, or a population of such cells.

In some embodiments, the cell, or population of cells, is a hepatocyte, a liver stellate cell, a Kupffer cell, or a population of such cells.

In some embodiments, the cell, or population of cells, is an osteoblast, an osteoclast, or a population of such cells.

In some embodiments, the cell, or population of cells, is an adipocyte, a preadipocyte, or a population of such cells.

In some embodiments, the cell, or population of cells, is a pancreatic islet cell (e.g., a beta cell, an alpha cell, or a delta cell), a pancreatic exocrine cell, or a population of such cells.

In some embodiments, the cell, or population of cells, is a Schwann cell, an oligodendrocyte, or a population of such cells.

In some embodiments, the cells are manipulated (e.g., converted or differentiated) from one cell type to another. In some embodiments, a pancreatic cell is manipulated into a beta islet cell. In some embodiments, a fibroblast is manipulated into an iPS cell. In some embodiments, a preadipocyte is manipulated into a brown fat cell. Other exemplary cells include, e.g., muscle cells, neural cells, leukocytes, and lymphocytes. In some embodiments, the cell being manipulated is selected from fibroblasts, monocytic precursors, B cells, exocrine cells, pancreatic progenitors, endocrine progenitors, hepatoblasts, myoblasts. In some embodiments, the cell is manipulated (e.g., converted or differentiated) into muscle.

In some embodiments, a nucleic acid at one or more target positions in a cell, or a population of cells, is altered e.g., as described herein. In some embodiments, a nucleic acid at one or more target positions in a cell, or a population of cells (e.g., target positions at one or more genes described herein) is altered, e.g., in vivo. In other embodiments, a nucleic acid at one or more target positions in a cell, or a population of cells (e.g., target positions at one or more genes described herein) is altered, e.g., ex vivo. The Cas9 molecule, nucleic acid template system, and/or gRNA molecules described herein can be delivered to a cell or to a population of cells.

In some embodiments, the cell, or the population of cells, is a T cell, a CD8⁺ T cell, a CD8⁺ naïve T cell, a central memory T cell, an effector memory T cell, a CD4⁺ T cell, a stem cell memory T cell, a helper T cell, a regulatory T cell, a cytotoxic T cell, a natural killer T cell, a hematopoietic stem cell, a long term hematopoietic stem cell, a short term hematopoietic stem cell, a multipotent progenitor cell, a lineage restricted progenitor cell, a lymphoid progenitor cell, a pancreatic progenitor cell, an endocrine progenitor cell, an exocrine progenitor cell, a myeloid progenitor cell, a common myeloid progenitor cell, an erythroid progenitor cell, a megakaryocyte erythroid progenitor cell, a monocytic precursor cell, an endocrine precursor cell, an exocrine cell, a fibroblast, a hepatoblast, a myoblast, a macrophage, an islet beta-cell, a cardiomyocyte, a blood cell, a ductal cell, an acinar cell, an alpha cell, a beta cell, a delta cell, a PP cell, a cholangiocyte, a retinal cell, a photoreceptor cell, a rod cell, a cone cell, a retinal pigmented epithelium cell, a trabecular meshwork cell, a cochlear hair cell, an outer hair cell, an inner hair cell, a pulmonary epithelial cell, a bronchial epithelial cell, an alveolar epithelial cell, a pulmonary epithelial progenitor cell, a striated muscle cell, a cardiac muscle cell, a muscle satellite cell, a myocyte, a neuron, a neuronal stem cell, a mesenchymal stem cell, an induced pluripotent stem (iPS) cell, an embryonic stem cell, a monocyte, a megakaryocyte, a neutrophil, an eosinophil, a basophil, a mast cell, a reticulocyte, a B cell, e.g. a progenitor B cell, a Pre B cell, a Pro B cell, a memory B cell, a plasma B cell, a gastrointestinal epithelial cell, a biliary epithelial cell, a pancreatic ductal epithelial cell, an intestinal stem cell, a hepatocyte, a liver stellate cell, a Kupffer cell, an osteoblast, an osteoclast, an adipocyte (e.g., a brown adipocyte, or a white adipocyte), a preadipocyte, a pancreatic precursor cell, a pancreatic islet cell, a pancreatic beta cell, a pancreatic alpha cell, a pancreatic delta cell, a pancreatic exocrine cell, a Schwann cell, or an oligodendrocyte, or a population of such cells.

In some embodiments, the cell, or the population of cells, is a mammalian cell, e.g., a human cell, a mouse cell, a rat cell, a sheep cell, a cow cell, a pig cell, a horse cell, a goat cell, a dog cell or a cat cell, or a population of mammalian cells. In one embodiment, the cell is a human cell.

In an embodiment, the cell, or population of cells, is manipulated ex vivo by altering a nucleic acid at one or more target positions, and administered to a subject. A cell, or population of cells, to be altered according to the methods disclosed herein, may include a stem cell such as, by way of example, an embryonic stem cell, an induced pluripotent stem cell or a neuronal stem cell, or a population of such cells. In an embodiment, the cell, or population of cells, is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, or a population of such cells, altered to correct a mutation and differentiated into a clinically relevant cell, or population of cells.

In some embodiments, the cell is a cell from a disease-causing organism, e.g., a bacterium, fungus, protozoan, or parasite. In some embodiments, the cell is a cell infected with a disease-causing organism (e.g., a virus, fungus, protozoan, or parasite).

In some embodiments, the cell is situated in the body of a subject. In such instances, the cell might be the subject's own cells or might be a cell of a disease-causing organism. In this case, a gRNA molecule, a Cas9 molecule, and a nucleic acid template system, may be administered to the subject as pharmaceutical compositions. In some embodiments, the subject is a mammal, e.g., a human, a farm animal (e.g., a cow, a pig, a horse, or a goat), or a companion animal (e.g., a dog or a cat).

In some embodiments, the subject suffers from a disease caused by a target position in a nucleic acid, e.g., a particular mutation, of a cell, or population of cells.

In some embodiments, the cell, or population of cells, is a diseased or mutant-bearing cell, or population of cells. Such cells can be altered to treat the disease, e.g., to correct a mutation, or to alter the phenotype of the cell, or population of cells, e.g., to inhibit the growth of a cancer cell or a population of cancer cells, e.g., a tumor. For example, a cell, or a population of cells, is associated with one or more diseases or conditions describe herein. In some embodiments, the cell is a cancer stem cell. For example, cancer stem cells can be manipulated by modulating the expression of one or more genes selected from TWIST (TF), HIF-1 alpha, HER2/neu, Snail (TF), or Wnt. In some embodiments, the cancer cell is selected from lung cancer cells, breast cancer cells, skin cancer cells, brain cancer cells, pancreatic cancer cells, hematopoietic cancer cells, liver cancer cells, kidney cancer cells, and ovarian cancer cells.

In some embodiments, the cell is characterized by a disorder caused by aberrant mtDNA. This disorder may be, e.g., a mtDNA depletion syndrome (e.g., Alpers or early infantile hepatocerebral syndromes) or a mtDNA deletion disorder (e.g., progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)).

In some embodiments, the cell, or population of cells, is a normal cell or a population of normal cells.

In some embodiments, the cell, or population of cells, is a stem cell or a progenitor cell (e.g., iPS, embryonic, hematopoietic, adipose, germline, lung, or neural stem or progenitor cells), or a population of such cells.

The cells may also be treated at a time when they are not situated in the body of a subject. In some embodiments, a cell, or a population of cells, is treated ex vivo to avoid exposing a patient to an agent or agents that cause undesirable side effects. In some embodiments, treating cells ex vivo allows a user to select a sub-population of cells to administer to the patient. The sub-population may be, e.g., cells having a nucleic acid that was successfully altered, or cells having a desired phenotype, such as minimal undesired alterations to DNA, or a phenotype that indicates the nucleic acid was successfully altered.

In some embodiments, the cell, or population of cells, is not situated in a subject's body and the cell, or population of cells, is modified for research or manufacturing purposes. In some embodiments, the cell, or population of cells, is suitable for producing a recombinant biological product. For example, the cell, or population of cells, can be a CHO cell or a fibroblast. In one embodiment, the cell, or population of cells, is a cell, or population of cells, that has been engineered to express a protein.

In some embodiments, the cell, or population of cells, is not actively dividing. In some embodiments, the cell is in in G0 phase (which is sometimes viewed as a prolonged G1 phase), is quiescent, or is senescent. In some embodiments, the population of cells are in G0 phase, are quiescent, or are senescent. In some embodiment, the quiescent cell can be a terminally differentiated cell. In some embodiments, the quiescent cell can be a neuron, a muscle cell, e.g., a cardiac muscle cell, a parenchymal cell e.g., a parenchymal liver or kidney cell, a hematopoietic cell e.g., a hematopoetic stem cell, a fibroblast, a stem cell e.g., an adult stem cell, a hepatic cell e.g., a hepatic stellate cell, an immune cell e.g., a T cell or B cell, or an epithelial cell. In some embodiments, the senescent cell may have shortened telomeres relative to an actively dividing cell, e.g., its telomeres may be of a length that triggers a halt in the cell cycle.

In some embodiments, the cell, or population of cells, is characterized by a disorder caused by aberrant mtDNA. This disorder may be, e.g., a mtDNA depletion syndrome (e.g., Alpers or early infantile hepatocerebral syndromes) or mtDNA deletion disorder (e.g., progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)).

In some embodiments, the cell, or population of cells, is actively dividing. In some embodiments, the cell is in G2 phase. In some embodiments, the population of cells comprises cells that are in G2 phase. In some embodiments, the cell is in G1 phase. In some embodiments, the population of cells comprises cells that are in G1 phase. In some embodiments, the cell is in S phase. In some embodiments, the population of cells comprises cells that are in S phase.

The technology described herein can be used to edit numerous types of genomes, including plant genomes. The CRISPR/Cas system has been used for plant genome editing, as has been described in, e.g., Belhaj et al., PLANT METHODS 9:39, 2013. Plant cells can carry out HDR, so a Cas9-induced nick or DSB can be repaired by HDR. Plant cells also have NHEJ machinery, and in some embodiments, NHEJ is inhibited, resulting in stimulation of HDR. Accordingly, in certain embodiments, the cell, or the population of cells, is a plant cell, e.g., a monocot plant cell, or a dicot plant cell, or a population of plant cell. In certain embodiments, the plant is a crop, e.g., a food crop. In certain embodiments, the plant is rice (e.g., Orzya sativa), maize (e.g., Zea mays), wheat (e.g., Triticum aestivum), soy (e.g., Glycine max), potato (e.g., Solanum tuberosum), a species of Nicotiana, a species of Arabidopsis e.g., Arabidopsis thaliana, cassava, sweet potato, sorghum, yam, plantain, or a citrus plant. In some embodiments, the plant is a pesticide-resistant plant, e.g., a plant that expresses one or more genes that confer resistance to a pesticide. In some embodiments, the plant is herbicide-resistant plant, e.g., a plant that expresses one or more genes that confer resistance to a herbicide. The herbicide may be, e.g., Roundup® (also known as glyphosate or N-(phosphonomethyl)glycine). In some embodiments, the plant produces a pesticide, e.g., Bt.

In some embodiments, the components used in the methods described herein (e.g., a Cas9 molecule and a gRNA) are introduced into the plant cell via protoplast transformation or agroinfiltration.

In some embodiments, after genome editing using the methods described herein, seeds are screened and a desired sub-population of seeds are selected. The sub-population may be, e.g., cells having a nucleic acid that was successfully altered, or cells having a desired phenotype such as minimal undesired alterations to DNA, or a phenotype that indicates the nucleic acid was successfully altered.

IX. Delivery, Formulations and Routes of Administration

The components, e.g., a Cas9 molecule and gRNA molecule can be delivered or formulated in a variety of forms, see, e.g., Tables IX.1-IX.3. When a Cas9 or gRNA component is encoded as DNA for delivery, the DNA will typically but not necessarily include a control region, e.g., comprising a promoter, to effect expression. Useful promoters for Cas9 molecule sequences include CMV, EF-1alpha, MSCV, PGK, CAG control promoters. Useful promoters for gRNAs include H1, EF-1alpha and U6 promoters. Promoters with similar or dissimilar strengths can be selected to tune the expression of components. Sequences encoding a Cas9 molecule can comprise a nuclear localization signal (NLS), e.g., an SV40 NLS. In an embodiment a promoter for a Cas9 molecule or a gRNA molecule can be, independently, inducible, tissue specific, or cell specific.

Table IX.1 provides non-limiting examples of the form in which the components can be delivered to a target cell. Merged cells indicate that the components of those columns are delivered via the same molecule, e.g., in the second row, the gRNA and the donor template nucleic acid are delivered on the same DNA molecule as indicated by the two merged cells.

TABLE IX.1 Elements Donor Tem- Cas9 gRNA plate Mole- Mole- Nucleic cule(s) cule(s) Acid Comments DNA DNA DNA In this embodiment, a Cas9 molecule, typically an eaCas9 molecule, and a gRNA are transcribed from DNA. In this embodiment, they are encoded on separate molecules. In this embodiment, the donor template is provided as a separate DNA molecule. DNA DNA In this embodiment, a Cas9 molecule, typically an eaCas9 molecule, and a gRNA are transcribed from DNA. In this embodiment, they are encoded on separate molecules. In this embodiment, the donor template is provided on the same DNA molecule that encodes the gRNA. DNA DNA In this embodiment, a Cas9 molecule, typically an eaCas9 molecule, and a gRNA are transcribed from DNA, here from a single molecule. In this embodiment, the donor template is provided as a separate DNA molecule. DNA DNA DNA In this embodiment, a Cas9 molecule, typically an eaCas9 molecule, and a gRNA are transcribed from DNA. In this embodiment, they are encoded on separate molecules. In this embodiment, the donor template is provided on the same DNA molecule that encodes the Cas9. DNA RNA DNA In this embodiment, a Cas9 molecule, typically an eaCas9 molecule, is transcribed from DNA, and a gRNA is provided as an RNA, e.g., as in vitro transcribed or synthesized RNA. In this embodiment, the donor template is provided as a separate DNA molecule. DNA RNA DNA In this embodiment, a Cas9 molecule, typically an eaCas9 molecule, is transcribed from DNA, and a gRNA is provided as an RNA, e.g., as in vitro transcribed or synthesized RNA. In this embodiment, the donor template is provided on the same DNA molecule that encodes the Cas9. mRNA RNA DNA In this embodiment, a Cas9 molecule, typically an eaCas9 molecule, is translated from in vitro transcribed or synthesized mRNA, and a gRNA is provided as an RNA, e.g., as in vitro transcribed or synthesized RNA. In this embodiment, the donor template is provided as a DNA molecule. mRNA DNA DNA In this embodiment, a Cas9 molecule, typically an eaCas9 molecule, is translated from in vitro transcribed or synthesized mRNA, and a gRNA is transcribed from DNA. In this embodiment, the donor template is provided as a separate DNA molecule. mRNA DNA In this embodiment, a Cas9 molecule, typically an eaCas9 molecule, is translated from in vitro transcribed or synthesized mRNA, and a gRNA is transcribed from DNA. In this embodiment, the donor template is provided on the same DNA molecule that encodes the gRNA. Protein DNA DNA In this embodiment, a Cas9 molecule, typically an eaCas9 molecule, is provided as a protein, and a gRNA is transcribed from DNA. In this embodiment, the donor template is provided as a separate DNA molecule. Protein DNA In this embodiment, a Cas9 molecule, typically an eaCas9 molecule, is provided as a protein, and a gRNA is transcribed from DNA. In this embodiment, the donor template is provided on the same DNA molecule that encodes the gRNA. Protein RNA DNA In this embodiment, an eaCas9 molecule is provided as a protein, and a gRNA is provided as transcribed or synthesized RNA. In this embodiment, the donor template is provided as a DNA molecule.

In some embodiments, a nucleic acid encoding an HDR-enhancer molecule is on a separate nucleic acid molecule from other components being delivered (e.g., gRNAs, Cas9 molecule, or template nucleic acid). In some embodiments, a DNA encoding an HDR-enhancer molecule is part of a DNA molecule encoding one or more, e.g., all, of a gRNA or a Cas9 molecule or comprising a template nucleic acid. In some embodiments, a DNA encoding an HDR-enhancer molecule is part of a DNA molecule listed in Table IX. 1.

Other delivery combinations are illustrated in Table IX.2

TABLE IX.2 Elements Donor Cas9 gRNA Template HDR- Molecule(s) Molecule(s) Nucleic Acid enhancer Comments DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA In this embodiment, the gene for the Cas9 molecule and the template nucleic acid are provided on the same DNA molecule. DNA DNA DNA DNA In this embodiment, the gene for the Cas9 molecule and the gene for the HDR enhancer are provided on the same DNA molecule. DNA DNA DNA DNA In this embodiment, the gene for the gRNA molecule and the template nucleic acid are provided on the same DNA molecule. DNA DNA DNA DNA In this embodiment, the template nucleic acid and the gene for the HDR enhancer are provided on the same DNA molecule. DNA DNA DNA DNA DNA DNA DNA DNA In this embodiment, the gene for the Cas9 molecule, the template nucleic acid, and the gene for the HDR-enhancer are provided on the same DNA molecule. DNA DNA DNA DNA In this embodiment, the gene for the Cas9 molecule, the template nucleic acid, the gene for the gRNA, and the gene for the HDR-enhancer are provided on the same DNA molecule. DNA DNA RNA DNA DNA In these embodiments, the Cas9 gene, template nucleic acid, and HDR enhancer can be provided on the same DNA molecule, on three different DNA molecules, or any two of the components can be on a first DNA molecule and the third component can be on another DNA molecule. DNA RNA DNA mRNA In these embodiments, the Cas9 gene and template nucleic acid can be on the same DNA molecule or on different DNA molecules. DNA RNA DNA Protein, small In these embodiments, the Cas9 molecule, or gene and template nucleic acid siRNA can be on the same DNA molecule or on different DNA molecules. mRNA RNA DNA DNA In these embodiments, the template nucleic acid and HDR- enhancer can be on the same DNA molecule or on different DNA molecules. mRNA RNA DNA mRNA mRNA RNA DNA Protein, small molecule, or siRNA mRNA DNA DNA DNA In these embodiments, the gRNA gene, template nucleic acid, and HDR enhancer gene can be provided on the same DNA molecule, on three different DNA molecules, or any two of the components can be on a first DNA molecule and the third component can be on another DNA molecule. mRNA DNA DNA mRNA In these embodiments, the gRNA gene and template nucleic acid can be on the same DNA molecule or on different DNA molecules. mRNA DNA DNA Protein, small In these embodiments, the gRNA molecule, or gene and template nucleic acid siRNA can be on the same DNA molecule or on different DNA molecules. Protein DNA DNA DNA In these embodiments, the gRNA gene, template nucleic acid, and HDR-enhancer gene can be provided on the same DNA molecule, on three different DNA molecules, or any two of the components can be on a first DNA molecule and the third component can be on another DNA molecule. Protein DNA DNA mRNA In these embodiments, the gRNA gene and template nucleic acid can be on the same DNA molecule or on different DNA molecules. Protein DNA DNA Protein, small In these embodiments, the gRNA molecule, or gene and template nucleic acid siRNA can be on the same DNA molecule or on different DNA molecules. Protein RNA DNA DNA In these embodiments, the template nucleic acid and HDR- enhancer gene can be on the same DNA molecule or on different DNA molecules. Protein RNA DNA mRNA Protein RNA DNA Protein, small molecule, or siRNA

Table IX.3 summarizes various delivery methods for the components of a Cas system, e.g., the Cas9 molecule component and the gRNA molecule component, as described herein.

TABLE IX.3 Delivery into Non- Duration Type of Dividing of Genome Molecule Delivery Vector/Mode Cells Expression Integration Delivered Physical (e.g., YES Transient NO Nucleic Acids electroporation, particle gun, and Proteins calcium phosphate transfection) Viral Retrovirus NO Stable YES RNA Lentivirus YES Stable YES/NO with RNA modifications Adenovirus YES Transient NO DNA Adeno- YES Stable NO DNA Associated Virus (AAV) Vaccinia Virus YES Transient NO DNA Herpes Simplex YES Stable NO DNA Virus Non-Viral Cationic YES Transient Depends on Nucleic Acids Liposomes what is and Proteins delivered Polymeric YES Transient Depends on Nucleic Acids Nanoparticles what is and Proteins delivered Biological Attenuated YES Transient NO Nucleic Acids Non-Viral Bacteria Delivery Engineered YES Transient NO Nucleic Acids Vehicles Bacteriophages Mammalian YES Transient NO Nucleic Acids Virus-like Particles Biological YES Transient NO Nucleic Acids liposomes: Erythrocyte Ghosts and Exosomes DNA-Based Delivery of a Cas9 Molecule and or a gRNA Molecule

DNA encoding Cas9 molecules (e.g., eaCas9 molecules), gRNA molecules, template nucleic acids, and/or HDR-enhancers, can be administered to subjects or delivered into cells by any appropriate method, e.g., by art-known methods or as described herein. For example, Cas9-encoding and/or gRNA-encoding DNA can be delivered, e.g., by vectors (e.g., viral or non-viral vectors), non-vector based methods (e.g., using naked DNA or DNA complexes), or a combination thereof.

In some embodiments, the nucleic acid, e.g., Cas9-, gRNA-, and/or HDR-enhancer-encoding DNA is delivered by a vector (e.g., viral vector/virus or plasmid).

In one embodiment, a vector can comprise a sequence that encodes a Cas9 molecule, a gRNA molecule, and/or an HDR-enhancer. In one embodiment, a vector can also comprise a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, mitochondrial localization), fused, e.g., to a Cas9 molecule sequence. For example, a vector can comprise a nuclear localization sequence (e.g., from SV40) fused to the sequence encoding the Cas9 molecule.

One or more regulatory/control elements, e.g., a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, internal ribosome entry sites (IRES), a 2A sequence, and splice acceptor or donor can be included in the vectors. In some embodiments, the promoter is recognized by RNA polymerase II (e.g., a CMV promoter).

In some embodiments, the vector or delivery vehicle is a viral vector (e.g., for generation of recombinant viruses). In some embodiments, the virus is a DNA virus (e.g., dsDNA or ssDNA virus). In other embodiments, the virus is an RNA virus (e.g., an ssRNA virus). Exemplary viral vectors/viruses include, e.g., retroviruses, lentiviruses, adenovirus, adeno-associated virus (AAV), vaccinia viruses, poxviruses, and herpes simplex viruses.

In some embodiments, the virus infects dividing cells. In other embodiments, the virus infects non-dividing cells. In some embodiments, the virus infects both dividing and non-dividing cells. In some embodiments, the virus can integrate into the host genome. In some embodiments, the virus is engineered to have reduced immunity, e.g., in human. In some embodiments, the virus is replication-competent. In other embodiments, the virus is replication-defective, e.g., having one or more coding regions for the genes necessary for additional rounds of virion replication and/or packaging replaced with other genes or deleted. In some embodiments, the virus causes transient expression of the Cas9 molecule and/or the gRNA molecule. In other embodiments, the virus causes long-lasting, e.g., at least 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, 2 years, or permanent expression, of the Cas9 molecule and/or the gRNA molecule. The packaging capacity of the viruses may vary, e.g., from at least about 4 kb to at least about 30 kb, e.g., at least about 5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30 kb, 35 kb, 40 kb, 45 kb, or 50 kb.

In some embodiments, the Cas9-, gRNA-, and/or HDR-enhancer-encoding DNA is delivered by a recombinant adenovirus. In some embodiments, the adenovirus is engineered to have reduced immunity in human.

In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a recombinant AAV. In some embodiments, the AAV can incorporate its genome into that of a host cell, e.g., a target cell as described herein. In some embodiments, the AAV is a self-complementary adeno-associated virus (scAAV), e.g., a scAAV that packages both strands which anneal together to form double stranded DNA. AAV serotypes that may be used in the disclosed methods, include AAV1, AAV2, modified AAV2 (e.g., modifications at Y444F, Y500F, Y730F and/or S662V), AAV3, modified AAV3 (e.g., modifications at Y705F, Y73iF and/or T492V), AAV4, AAV5, AAV6, modified AAV6 (e.g., modifications at S663V and/or T492V), AAV8, AAV 8.2, AAV9, AAV rhl0, and pseudotyped AAV, such as AAV2/8, AAV2/5 and AAV2/6 can also be used in the disclosed methods.

In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a non-vector based method (e.g., using naked DNA or DNA complexes). For example, the DNA can be delivered, e.g., by organically modified silica or silicate (Ormosil), electroporation, gene gun, sonoporation, magnetofection, lipid-mediated transfection, dendrimers, inorganic nanoparticles, calcium phosphates, or a combination thereof.

In an embodiment, the delivery vehicle is a non-viral vector. In an embodiment, the non-viral vector is an inorganic nanoparticle. Exemplary inorganic nanoparticles include, e.g., magnetic nanoparticles (e.g., Fe₃MnO₂) and silica. The outer surface of the nanoparticle can be conjugated with a positively charged polymer (e.g., polyethylenimine, polylysine, polyserine) which allows for attachment (e.g., conjugation or entrapment) of payload. In an embodiment, the non-viral vector is an organic nanoparticle (e.g., entrapment of the payload inside the nanoparticle). Exemplary organic nanoparticles include, e.g., SNALP liposomes that contain cationic lipids together with neutral helper lipids which are coated with polyethylene glycol (PEG) and protamine and nucleic acid complex coated with lipid coating.

Exemplary lipids for gene transfer are shown below in Table IX.4.

TABLE IX.4 Lipids Used for Gene Transfer Lipid Abbreviation Feature 1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine DOPC Helper 1,2-Dioleoyl-sn-glycero-3- DOPE Helper phosphatidylethanolamine Cholesterol Helper N-[1-(2,3-Dioleyloxy)prophyl]N,N,N- DOTMA Cationic trimethylammonium chloride 1,2-Dioleoyloxy-3-trimethylammonium-propane DOTAP Cationic Dioctadecylamidoglycylspermine DOGS Cationic N-(3-Aminopropyl)-N,N-dimethyl-2,3- GAP-DLRIE Cationic bis(dodecyloxy)-1-propanaminium bromide Cetyltrimethylammonium bromide CTAB Cationic 6-Lauroxyhexyl ornithinate LHON Cationic 1-(2,3-Dioleoyloxypropyl)-2,4,6- 2Oc Cationic trimethylpyridinium 2,3-Diolcyloxy-N-[2(sperminecarboxamido- DOSPA Cationic ethyl]-N,N-dimethyl-1- propanaminium trifluoroacetate 1,2-Dioleyl-3-trimethylammonium-propane DOPA Cationic N-(2-Hydroxyethyl)-N,N-dimethyl-2,3- MDRIE Cationic bis(tetradecyloxy)-1-propanaminium bromide Dimyristooxypropyl dimethyl hydroxyethyl DMRI Cationic ammonium bromide 3β-[N-(N′,N′-Dimethylaminoethane)- DC-Chol Cationic carbamoyl]cholesterol Bis-guanidium-tren-cholesterol BGTC Cationic 1,3-Diodeoxy-2-(6-carboxy-spermyl)-propylamide DOSPER Cationic Dimethyloctadecylammonium bromide DDAB Cationic Dioctadecylamidoglicylspermidin DSL Cationic rac-[(2,3-Dioctadecyloxypropyl)(2- CLIP-1 Cationic hydroxyethyl)]-dimethylammonium chloride rac-[2(2,3-Dihexadecyloxypropyl- CLIP-6 Cationic oxymethyloxy)ethyl]trimethylammonium bromide Ethyldimyristoylphosphatidylcholine EDMPC Cationic 1,2-Distcaryloxy-N,N-dimethyl-3-aminopropane DSDMA Cationic 1,2-Dimyristoyl-trimethylammonium propane DMTAP Cationic O,O′-Dimyristyl-N-lysyl aspartate DMKE Cationic 1,2-Distearoyl-sn-glycero-3-ethylphosphocholine DSEPC Cationic N-Palmitoyl D-erythro-sphingosyl carbamoyl- CCS Cationic spermine N-t-Butyl-N0-tetradecyl-3- diC14- Cationic tetradecylaminopropionamidine amidine Octadecenolyoxy[ethyl-2-heptadecenyl- DOTIM Cationic 3 hydroxyethyl] imidazolinium chloride N1-Cholesteryloxycarbonyl-3,7-diazanonane- CDAN Cationic 1,9-diamine 2-(3-[Bis(3-amino-propyl)- RPR209120 Cationic amino]propylamino)-N- ditetradecylcarbamoylme-ethyl-acetamide 1,2-dilinoleyloxy-3-dimethylaminopropane DLinDMA Cationic 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]- DLin-KC2- Cationic dioxolane DMA dilinoleyl-methyl-4-dimethylaminobutyrate DLin-MC3- Cationic DMA

Exemplary polymers for gene transfer are shown below in Table IX.5.

TABLE IX.5 Polymers Used for Gene Transfer Polymer Abbreviation Poly(ethylene)glycol PEG Polyethylenimine PEI Dithiobis(succinimidylpropionate) DSP Dimethyl-3,3′-dithiobispropionimidate DTBP Poly(ethylene imine)biscarbamate PEIC Poly(L-lysine) PLL Histidine modified PLL Poly(N-vinylpyrrolidone) PVP Poly(propylenimine) PPI Poly(amidoamine) PAMAM Poly(amidoethylenimine) SS-PAEI Triethylenetetramine TETA Poly(β-aminoester) Poly(4-hydroxy-L-proline ester) PHP Poly(allylamine) Poly(α-[4-aminobutyl]-L-glycolic acid) PAGA Poly(D,L-lactic-co-glycolic acid) PLGA Poly(N-ethyl-4-vinylpyridinium bromide) Poly(phosphazene)s PPZ Poly(phosphoester)s PPE Poly(phosphoramidate)s PPA Poly(N-2-hydroxypropylmethacrylamide) pHPMA Poly (2-(dimethylamino)ethyl methacrylate) pDMAEMA Poly(2-aminoethyl propylene phosphate) PPE-EA Chitosan Galactosylated chitosan N-Dodacylated chitosan Histone Collagen Dextran-spermine D-SPM

In an embodiment, the vehicle has targeting modifications to increase target cell update of nanoparticles and liposomes, e.g., cell specific antigens, monoclonal antibodies, single chain antibodies, aptamers, polymers, sugars, and cell penetrating peptides. In an embodiment, the vehicle uses fusogenic and endosome-destabilizing peptides/polymers. In an embodiment, the vehicle undergoes acid-triggered conformational changes (e.g., to accelerate endosomal escape of the cargo). In an embodiment, a stimuli-cleavable polymer is used, e.g., for release in a cellular compartment. For example, disulfide-based cationic polymers that are cleaved in the reducing cellular environment can be used.

In an embodiment, the delivery vehicle is a biological non-viral delivery vehicle.

In an embodiment, one or more nucleic acid molecules (e.g., a DNA molecule or a template nucleic acid) other than the components of a Cas system, e.g., the Cas9 molecule component and/or the gRNA molecule component described herein, are delivered. In an embodiment, the nucleic acid molecule is delivered at the same time as one or more of the components of the Cas system are delivered. In an embodiment, the nucleic acid molecule is delivered before or after (e.g., less than about 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 4 weeks) one or more of the components of the Cas system are delivered. In an embodiment, the nucleic acid molecule is delivered by a different means than one or more of the components of the Cas system, e.g., the Cas9 molecule component and/or the gRNA molecule component, are delivered. The nucleic acid molecule can be delivered by any of the delivery methods described herein. For example, the nucleic acid molecule can be delivered by a viral vector, e.g., an integration-deficient lentivirus, and the Cas9 molecule component and/or the gRNA molecule component can be delivered by electroporation, e.g., such that the toxicity caused by nucleic acids (e.g., DNAs) can be reduced. In an embodiment, the nucleic acid molecule encodes a therapeutic protein, e.g., a protein described herein. In an embodiment, the nucleic acid molecule encodes an RNA molecule, e.g., an RNA molecule described herein. In some embodiments, the nucleic acid is a template nucleic acid capable of participating in HDR.

Delivery of RNA Encoding a Cas9 Molecule

RNA encoding Cas9 molecules (e.g., eaCas9 molecules) and/or gRNA molecules, can be delivered into cells, e.g., target cells described herein, by any appropriate method, including art-known methods or methods described herein. For example, Cas9-encoding and/or gRNA-encoding RNA can be delivered, e.g., by microinjection, electroporation, lipid-mediated transfection, peptide-mediated delivery, or a combination thereof.

Delivery of Cas9 Molecule Protein

Cas9 molecules (e.g., eaCas9 molecules) can be delivered into cells by any appropriate method, including art-known methods or methods described herein. For example, Cas9 protein molecules can be delivered, e.g., by microinjection, electroporation, lipid-mediated transfection, peptide-mediated delivery, or a combination thereof. Delivery can be accompanied by DNA encoding a gRNA or by a gRNA.

Route of Administration

Systemic modes of administration include oral and parenteral routes. Parenteral routes include, by way of example, intravenous, intrarterial, intraosseous, intramuscular, intradermal, subcutaneous, intranasal and intraperitoneal routes. Components administered systemically may be modified or formulated to target the components to the desired cell type.

Local modes of administration include, by way of example, intrathecal, intracerebroventricular, intraparenchymal (e.g., localized intraparenchymal delivery to the striatum (e.g., into the caudate or into the putamen)), cerebral cortex, precentral gyrus, hippocampus (e.g., into the dentate gyrus or CA3 region), temporal cortex, amygdala, frontal cortex, thalamus, cerebellum, medulla, hypothalamus, tectum, tegmentum or substantia nigra intraocular, intraorbital, subconjuctival, intravitreal, subretinal or transscleral routes. In an embodiment, significantly smaller amounts of the components (compared with systemic approaches) may exert an effect when administered locally (for example, intraparenchymal or intravitreal) compared to when administered systemically (for example, intravenously). Local modes of administration can reduce or eliminate the incidence of potentially toxic side effects that may occur when therapeutically effective amounts of a component are administered systemically.

Administration may be provided as a periodic bolus or as continuous infusion from an internal reservoir or from an external reservoir (for example, from an intravenous bag). Components may be administered locally, for example, by continuous release from a sustained release drug delivery device.

In some embodiments, the methods herein involve delivering macromolecules (e.g., a Cas9 protein or nucleic acid, a gRNA or nucleic acid encoding a gRNA, or a template nucleic acid, or any combination thereof) and small molecules (e.g., an HDR-enhancer). In some embodiments, the small molecule is delivered via a different route from one or more macromolecules. The small molecule may be administered, for instance, in a form suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, and intraarticular), inhalation (including fine particle dusts or mists which may be generated by means of various types of metered doses, pressurized aerosols, nebulizers or insufflators, and including intranasally or via the lungs), rectal and topical (including dermal, transdermal, transmucosal, buccal, sublingual, and intraocular) administration, although the most suitable route may depend upon, for example, the condition and disorder of the recipient.

Bi-Modal or Differential Delivery of Components

Separate delivery of the components of a Cas system, e.g., the Cas9 molecule component and the gRNA molecule component, and more particularly, delivery of the components by differing modes, can enhance performance, e.g., by improving tissue specificity and safety.

In an embodiment, the Cas9 molecule and the gRNA molecule are delivered by different modes, or as sometimes referred to herein as differential modes. Different or differential modes, as used herein, refer to modes of delivery that confer different pharmacodynamic or pharmacokinetic properties on the subject component molecule, e.g., a Cas9 molecule, gRNA molecule, or template nucleic acid. For example, the modes of delivery can result in different tissue distribution, different half-life, or different temporal distribution, e.g., in a selected compartment, tissue, or organ. In many embodiments, the components are delivered so that Cas9 and the gRNA will be present in the same cell at the same time. In some embodiments, the HDR-enhancer molecule is delivered in a manner that allows HDR to be the favored repair pathway at the time the Cas9 molecule and gRNA cleave the desired region of the genome.

In some embodiments, two gRNAs are delivered to a cell so that a first nickase will make a first single strand break and a second nickase will make a second single strand break. In such embodiments, the two gRNAs and other components (e.g., the Cas9 molecule) are delivered such that the two breaks are made at substantially the same time. In some embodiments this comprises the second break being formed before the first break engages with machinery specific to the SSBR (single strand break repair) pathway, and in some embodiments, it comprises the second break being formed before the first break is repaired. More generally, when one desires to make two or more breaks in a target nucleic acid, the gRNAs and other components can be delivered such that the two or more breaks are made at substantially the same time.

Some modes of delivery, e.g., delivery by a nucleic acid vector that persists in a cell, or in progeny of a cell, e.g., by autonomous replication or insertion into cellular nucleic acid, result in more persistent expression of and presence of a component. Examples include viral, e.g., adeno-associated virus or lentivirus, delivery.

By way of example, the components, e.g., a Cas9 molecule and a gRNA molecule, can be delivered by modes that differ in terms of resulting half-life or persistent of the delivered component the body, or in a particular compartment, tissue or organ. In an embodiment, a gRNA molecule can be delivered by such modes. The Cas9 molecule component can be delivered by a mode which results in less persistence or less exposure to the body or a particular compartment or tissue or organ.

More generally, in an embodiment, a first mode of delivery is used to deliver a first component and a second mode of delivery is used to deliver a second component. The first mode of delivery confers a first pharmacodynamic or pharmacokinetic property. The first pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ. The second mode of delivery confers a second pharmacodynamic or pharmacokinetic property. The second pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ.

In an embodiment, the first pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure, is more limited than the second pharmacodynamic or pharmacokinetic property.

In an embodiment, the first mode of delivery is selected to optimize, e.g., minimize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure.

In an embodiment, the second mode of delivery is selected to optimize, e.g., maximize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure.

In an embodiment, the first mode of delivery comprises the use of a relatively persistent element, e.g., a nucleic acid, e.g., a plasmid or viral vector, e.g., an AAV or lentivirus. As such vectors are relatively persistent product transcribed from them would be relatively persistent.

In an embodiment, the second mode of delivery comprises a relatively transient element, e.g., an RNA or protein.

In an embodiment, the first component comprises gRNA, and the delivery mode is relatively persistent, e.g., the gRNA is transcribed from a plasmid or viral vector, e.g., an AAV or lentivirus. Transcription of these genes would be of little physiological consequence because the genes generally do not encode for a protein product, and the gRNAs are incapable of acting in isolation. The second component, a Cas9 molecule, is delivered in a transient manner, for example as mRNA or as protein, ensuring that the full Cas9 molecule/gRNA molecule complex is only present and active for a short period of time.

Furthermore, the components can be delivered in different molecular form or with different delivery vectors that complement one another to enhance safety and tissue specificity.

Use of differential delivery modes can enhance performance, safety and efficacy. For example, the likelihood of an eventual off-target modification can be reduced. Delivery of immunogenic components, e.g., Cas9 molecules, by less persistent modes can reduce immunogenicity, as peptides from the bacterially-derived Cas enzyme are displayed on the surface of the cell by MHC molecules. A two-part delivery system can alleviate these drawbacks.

Differential delivery modes can be used to deliver components to different, but overlapping target regions. The formation active complex is minimized outside the overlap of the target regions. Thus, in an embodiment, a first component, e.g., a gRNA molecule is delivered by a first delivery mode that results in a first spatial, e.g., tissue, distribution. A second component, e.g., a Cas9 molecule is delivered by a second delivery mode that results in a second spatial, e.g., tissue, distribution. In an embodiment, the first mode comprises a first element selected from a liposome, nanoparticle, e.g., polymeric nanoparticle, and a nucleic acid, e.g., viral vector. The second mode comprises a second element selected from the group. In an embodiment, the first mode of delivery comprises a first targeting element, e.g., a cell specific receptor or an antibody, and the second mode of delivery does not include that element. In embodiment, the second mode of delivery comprises a second targeting element, e.g., a second cell specific receptor or second antibody.

When the Cas9 molecule is delivered in a virus delivery vector, a liposome, or polymeric nanoparticle, there is the potential for delivery to and therapeutic activity in multiple tissues, when it may be desirable to only target a single tissue. A two-part delivery system can resolve this challenge and enhance tissue specificity. If the gRNA molecule and the Cas9 molecule are packaged in separated delivery vehicles with distinct but overlapping tissue tropism, the fully functional complex is only formed in the tissue that is targeted by both vectors.

Ex Vivo Delivery

In some embodiments, components described in Table IX.1 or IX.2 and a HDR-enhancer are introduced into cells which are then introduced into the subject. Methods of introducing the components can include, e.g., any of the delivery methods described in Table IX.3.

In some embodiments, treating cells with an HDR-enhancer ex vivo reduces the risk of that HDR-enhancer causing a side effect that would likely occur if the HDR-enhancer was administered to the patient's body.

In some embodiments, the cells are contacted with a Cas9 molecule (or a nucleic acid encoding it) ex vivo. In some embodiments, the cells are contacted with a gRNA (or a nucleic acid encoding it) ex vivo. In some embodiment, the cells are contacted with a template nucleic acid ex vivo. In some embodiments, the cells are contacted with an HDR-enhancer (or a nucleic acid encoding it) ex vivo. In some embodiments, the cells are contacted with two, three, or all four of the preceding compositions (or nucleic acids encoding them) ex vivo. In some embodiments, the cells are contacted with one or more of the preceding components (or nucleic acids encoding them), and one or more remaining components are administered to the patient.

X. Modified Nucleosides, Nucleotides, and Nucleic Acids

Modified nucleosides and modified nucleotides can be present in nucleic acids, e.g., particularly gRNA and/or a template nucleic acid, but also other forms of DNA or RNA, e.g., mRNA, RNAi, or siRNA. As described herein, “nucleoside” is defined as a compound containing a five-carbon sugar molecule (a pentose such as deoxyribose or ribose) or derivative thereof, and an organic base (purine or pyrimidine, or a derivative thereof). As described herein, “nucleotide” is defined as a nucleoside further comprising a phosphate group.

Modified nucleosides and nucleotides can include one or more of:

-   -   (i) alteration, e.g., replacement, of one or both of the         non-linking phosphate oxygens and/or of one or more of the         linking phosphate oxygens in the phosphodiester backbone         linkage;     -   (ii) alteration, e.g., replacement, of a constituent of the         ribose or deoxyribose sugar, e.g., of the 2′ hydroxyl on the         sugar;     -   (iii) wholesale replacement of the phosphate moiety with         “dephospho” linkers;     -   (iv) modification or replacement of a naturally occurring         nucleobase;     -   (v) replacement or modification of the ribose- or         deoxyribose-phosphate backbone;     -   (vi) modification of the 3′ end or 5′ end of the         oligonucleotide, e.g., removal, modification or replacement of a         terminal phosphate group or conjugation of a moiety; and     -   (vii) modification of the sugar.

The modifications listed above can be combined to provide modified nucleosides and nucleotides that can have two, three, four, or more modifications. For example, a modified nucleoside or nucleotide can have a modified sugar and a modified nucleobase. In an embodiment, every base of a gRNA or template nucleic acid is modified, e.g., all bases have a modified phosphate group, e.g., all are phosphorothioate groups. In an embodiment, all, or substantially all, of the phosphate groups of a unimolecular or modular gRNA molecule or template nucleic acid are replaced with phosphorothioate groups.

In an embodiment, modified nucleotides, e.g., nucleotides having modifications as described herein, can be incorporated into a nucleic acid, e.g., a “modified nucleic acid.” In some embodiments, the modified nucleic acids comprise one, two, three or more modified nucleotides. In some embodiments, at least 5% (e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%) of the positions in a modified nucleic acid are a modified nucleotides.

Unmodified nucleic acids can be prone to degradation by, e.g., cellular nucleases. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Accordingly, in one aspect the modified nucleic acids described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability toward nucleases.

In some embodiments, the modified nucleosides, modified nucleotides, and modified nucleic acids described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo. The term “innate immune response” includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, generally of viral or bacterial origin, which involves the induction of cytokine expression and release, particularly the interferons, and cell death. In some embodiments, the modified nucleosides, modified nucleotides, and modified nucleic acids described herein can disrupt binding of a major groove interacting partner with the nucleic acid. In some embodiments, the modified nucleosides, modified nucleotides, and modified nucleic acids described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo, and also disrupt binding of a major groove interacting partner with the nucleic acid.

In some embodiments, a template nucleic acid comprises modifications, e.g., modified nucleotides, modifications to the backbone, and other modifications described herein. In some embodiments, the modification improves the stability of the template nucleic acid, e.g., by increasing its resistance to endonucleases and/or exonucleases.

In some embodiments, a template nucleic acid that comprises modifications is double stranded, e.g., is double stranded DNA. In some such embodiments, all the modifications are confined to one strand. In other embodiments, modifications are present on both strands. Modifications may be present in the 5′ homology arm, the 3′ homology arm, or the replacement sequence, or any combination thereof. In some embodiments, modifications are present in one or both homology arms but not the replacement sequence.

In some embodiments, a template nucleic acid that comprises modifications is single stranded, e.g., is single stranded DNA.

Definitions of Chemical Groups

As used herein, “alkyl” is meant to refer to a saturated hydrocarbon group which is straight-chained or branched. Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. An alkyl group can contain from 1 to about 20, from 2 to about 20, from 1 to about 12, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms.

As used herein, “aryl” refers to monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbons such as, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, aryl groups have from 6 to about 20 carbon atoms.

As used herein, “alkenyl” refers to an aliphatic group containing at least one double bond.

As used herein, “alkynyl” refers to a straight or branched hydrocarbon chain containing 2-12 carbon atoms and characterized in having one or more triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, propargyl, and 3-hexynyl.

As used herein, “arylalkyl” or “aralkyl” refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of “arylalkyl” or “aralkyl” include benzyl, 2-phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl, and trityl groups.

As used herein, “cycloalkyl” refers to a cyclic, bicyclic, tricyclic, or polycyclic non-aromatic hydrocarbon groups having 3 to 12 carbons. Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclopentyl, and cyclohexyl.

As used herein, “heterocyclyl” refers to a monovalent radical of a heterocyclic ring system. Representative heterocyclyls include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, and morpholinyl.

As used herein, “heteroaryl” refers to a monovalent radical of a heteroaromatic ring system. Examples of heteroaryl moieties include, but are not limited to, imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrrolyl, furanyl, indolyl, thiophenyl pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolizinyl, purinyl, naphthyridinyl, quinolyl, and pteridinyl.

Phosphate Backbone Modifications

The Phosphate Group

In some embodiments, the phosphate group of a modified nucleotide can be modified by replacing one or more of the oxygens with a different substituent. Further, the modified nucleotide, e.g., modified nucleotide present in a modified nucleic acid, can include the wholesale replacement of an unmodified phosphate moiety with a modified phosphate as described herein. In some embodiments, the modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.

Examples of modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphate backbone moiety can be replaced by any of the following groups: sulfur (S), selenium (Se), BR₃ (wherein R can be, e.g., hydrogen, alkyl, or aryl), C (e.g., an alkyl group, an aryl group, and the like), H, NR₂ (wherein R can be, e.g., hydrogen, alkyl, or aryl), or OR (wherein R can be, e.g., alkyl or aryl). The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral; that is to say that a phosphorous atom in a phosphate group modified in this way is a stereogenic center. The stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp).

Phosphorodithioates have both non-bridging oxygens replaced by sulfur. The phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligoribonucleotide (or oligodeoxyribonucleotide) diastereomers. In some embodiments, modifications to one or both non-bridging oxygens can also include the replacement of the non-bridging oxygens with a group independently selected from S, Se, B, C, H, N, and OR (R can be, e.g., alkyl or aryl).

The phosphate linker can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens.

Replacement of the Phosphate Group

The phosphate group can be replaced by non-phosphorus containing connectors. In some embodiments, the charge phosphate group can be replaced by a neutral moiety.

Examples of moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.

Replacement of the Ribophosphate Backbone

Scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. In some embodiments, the nucleobases can be tethered by a surrogate backbone. Examples can include, without limitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.

Sugar Modifications

The modified nucleosides and modified nucleotides can include one or more modifications to the sugar group. For example, the 2′ hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents. In some embodiments, modifications to the 2′ hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2′-alkoxide ion. The 2′-alkoxide can catalyze degradation by intramolecular nucleophilic attack on the linker phosphorus atom.

Examples of “oxy”-2′ hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein “R” can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); polyethyleneglycols (PEG), O(CH₂CH₂O),CH₂CH₂OR wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). In some embodiments, the “oxy”-2′ hydroxyl group modification can include “locked” nucleic acids (LNA) in which the 2′ hydroxyl can be connected, e.g., by a C₁₋₆ alkylene or C₁₋₆ heteroalkylene bridge, to the 4′ carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; O-amino (wherein amino can be, e.g., NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, O(CH₂)_(n)-amino, (wherein amino can be, e.g., NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the “oxy”-2′ hydroxyl group modification can include the methoxyethyl group (MOE), (OCH₂CH₂OCH₃, e.g., a PEG derivative).

“Deoxy” modifications can include hydrogen (i.e. deoxyribose sugars, e.g., at the overhang portions of partially ds RNA); halo (e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH₂CH₂NH)_(n)CH₂CH₂-amino (wherein amino can be, e.g., as described herein), —NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with, e.g., an amino as described herein.

The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar. The nucleotide “monomer” can have an alpha linkage at the 1′ position on the sugar, e.g., alpha-nucleosides. The modified nucleic acids can also include “abasic” sugars, which lack a nucleobase at C-1′. These abasic sugars can also be further modified at one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that are in the L form, e.g., L-nucleosides.

Generally, RNA includes the sugar group ribose, and DNA includes the sugar group deoxyribose, each of which is a 5-membered ring having an oxygen. Exemplary modified nucleosides and modified nucleotides can include, without limitation, replacement of the oxygen in the ribose or deoxyribose ring (e.g., with sulfur (S), selenium (Se), or alkylene, such as, e.g., methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for example, anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone). In some embodiments, the modified nucleotides can include multicyclic forms (e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), threose nucleic acid (TNA, where ribose is replaced with α-L-threofuranosyl-(3′→2′)).

Modifications on the Nucleobase

The modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified nucleic acid, can include a modified nucleobase. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or wholly replaced to provide modified nucleosides and modified nucleotides that can be incorporated into modified nucleic acids. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine or pyrimidine analog. In some embodiments, the nucleobase can include, for example, naturally-occurring and synthetic derivatives of a base.

Uracil

In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include without limitation pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m³U), 5-methoxy-uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine 5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U), 5-methoxycarbonylmethyl-uridine (mcm⁵U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s2U), 5-aminomethyl-2-thio-uridine (nm⁵s2U), 5-methylaminomethyl-uridine (mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s2U), 5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U), 5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine (cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τcm⁵U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(rm⁵s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m¹ψ), 5-methyl-2-thio-uridine (m⁵s2U), 1-methyl-4-thio-pseudouridine (m¹s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp³U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ψ), 5-(isopentenylaminomethyl)uridine (inm⁵U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s2U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um), 2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2′-O-methyl-uridine (mem ⁵Um), 5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um), 5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um), 3,2′-O-dimethyl-uridine (m³Um), 5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine, deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-(1-E-propenylamino)uridine, pyrazolo[3,4-d]pyrimidines, xanthine, and hypoxanthine.

Thymine

In some embodiments, the modified nucleobase is a modified thymine. Thymine differs from uracil in that thymine has a methyl group on carbon 5 of the 6-carbon ring, while uracil has a hydrogen in that position. In some embodiments, the modified thymine is derived from one of the modified uracils described in the previous paragraph, but having said methyl group instead of a hydrogen.

Cytosine

In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include without limitation 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m³C), N4-acetyl-cytidine (act), 5-formyl-cytidine (f⁵C), N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k²C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm), 5,2′-O-dimethyl-cytidine (m⁵Cm), N4-acetyl-2′-O-methyl-cytidine (ac⁴Cm), N4,2′-O-dimethyl-cytidine (m⁴Cm), 5-formyl-2′-O-methyl-cytidine (f⁵Cm), N4,N4,2′-O-trimethyl-cytidine (m⁴2Cm), 1-thio-cytidine, 2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.

Adenine

In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include without limitation 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m¹A), 2-methyl-adenine (m²A), N6-methyl-adenosine (m⁶A), 2-methylthio-N6-methyl-adenosine (ms2 m⁶A), N6-isopentenyl-adenosine (i⁶A), 2-methylthio-N6-isopentenyl-adenosine (ms²i⁶A), N6-(cis-hydroxyisopentenyl)adenosine (io⁶A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io⁶A), N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine (t⁶A), N6-methyl-N6-threonylcarbamoyl-adenosine (m⁶t⁶A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms²g⁶A), N6,N6-dimethyl-adenosine (m⁶2A), N6-hydroxynorvalylcarbamoyl-adenosine (hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn⁶A), N6-acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am), N⁶,2′-O-dimethyl-adenosine (m⁶Am), N⁶-Methyl-2′-deoxyadenosine, N6,N6,2′-O-trimethyl-adenosine (m⁶2Am), 1,2′-O-dimethyl-adenosine (m¹Am), 2′-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2′-F-ara-adenosine, 2′-F-adenosine, 2′-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

Guanine

In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include without limitation inosine (I), 1-methyl-inosine (m¹I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o₂yW), hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ₀), 7-aminomethyl-7-deaza-guanosine (preQ₁), archaeosine (G⁺), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m⁷G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m′G), N2-methyl-guanosine (m²G), N2,N2-dimethyl-guanosine (m² 2G), N2,7-dimethyl-guanosine (m²,7G), N2, N2,7-dimethyl-guanosine (m²,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-meth thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2′-O-methyl-guanosine (Gm), N2-methyl-2′-O-methyl-guanosine (m²Gm), N2,N2-dimethyl-2′-O-methyl-guanosine (m² 2Gm), 1-methyl-2′-O-methyl-guanosine (m′Gm), N2,7-dimethyl-2′-O-methyl-guanosine (m²,7Gm), 2′-O-methyl-inosine (Im), 1,2′-O-dimethyl-inosine (m′Im), O⁶-phenyl-2′-deoxyinosine, 2′-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, O⁶-methyl-guanosine, O⁶-Methyl-2′-deoxyguanosine, 2′-F-ara-guanosine, and 2′-F-guanosine.

Modified gRNAs

In some embodiments, the modified nucleic acids can be modified gRNAs. In some embodiments, gRNAs can be modified at the 3′ end. In this embodiment, the gRNAs can be modified at the 3′ terminal U ribose. For example, the two terminal hydroxyl groups of the U ribose can be oxidized to aldehyde groups and a concomitant opening of the ribose ring to afford a modified nucleoside as shown below:

wherein “U” can be an unmodified or modified uridine.

In another embodiment, the 3′ terminal U can be modified with a 2′3′ cyclic phosphate as shown below:

wherein “U” can be an unmodified or modified uridine.

In some embodiments, the gRNA molecules may contain 3′ nucleotides which can be stabilized against degradation, e.g., by incorporating one or more of the modified nucleotides described herein. In this embodiment, e.g., uridines can be replaced with modified uridines, e.g., 5-(2-amino)propyl uridine, and 5-bromo uridine, or with any of the modified uridines described herein; adenosines and guanosines can be replaced with modified adenosines and guanosines, e.g., with modifications at the 8-position, e.g., 8-bromo guanosine, or with any of the modified adenosines or guanosines described herein. In some embodiments, deaza nucleotides, e.g., 7-deaza-adenosine, can be incorporated into the gRNA. In some embodiments, O- and N-alkylated nucleotides, e.g., N6-methyl andenosine, can be incorporated into the gRNA. In some embodiments, sugar-modified ribonucleotides can be incorporated, e.g., wherein the 2′ OH-group is replaced by a group selected from H, —OR, —R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), halo, —SH, —SR (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), amino (wherein amino can be, e.g., NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or cyano (—CN). In some embodiments, the phosphate backbone can be modified as described herein, e.g., with a phosphothioate group. In some embodiments, the nucleotides in the overhang region of the gRNA can each independently be a modified or unmodified nucleotide including, but not limited to 2′-sugar modified, such as, 2-F 2′-O-methyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine (Teo), 2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinations thereof.

In an embodiment, one or more or all of the nucleotides in single stranded RNA molecule, e.g., a gRNA molecule, are deoxynucleotides.

In another aspect, methods and compositions discussed herein provide methods and compositions for gene editing by using a gRNA molecule which comprises a polyA tail. In one embodiment, a polyA tail of undefined length ranging from 1 to 1000 nucleotide(s) is added enzymatically using a polymerase such as E. coli polyA polymerase (E-PAP). In one embodiment, the polyA tail of a specified length (e.g., 1, 5, 10, 20, 30, 40, 50, 60, 100, or 150 nucleotides (SEQ ID NO: 2289)) is encoded on a DNA template and transcribed with the gRNA via an RNA polymerase (e.g., T7 RNA polymerase). In one embodiment, a polyA tail of defined length (e.g., 1, 5, 10, 20, 30, 40, 50, 60, 100, or 150 nucleotides (SEQ ID NO: 2289)) is synthesized as a synthetic oligonucleotide and ligated on the 3′ end of the gRNA with either an RNA ligase or a DNA ligase with our without a splinted DNA oligonucleotide complementary to the guide RNA and the polyA oligonucleotide. In one embodiment, the entire gRNA including a defined length of polyA tail is made synthetically, in one or several pieces, and, if made in more than one piece, ligated together by either an RNA ligase or a DNA ligase with or without a splinted oligonucleotide.

Modified Template Nucleic Acids

In some embodiments, the template nucleic acid comprises chemical modifications. These modifications may, e.g., increase the stability or half-life of the nucleic acid or reduce the innate immune response to the nucleic acid.

In some embodiments, the template nucleic acid can be modified at one or two 3′ ends. In this embodiment, the template nucleic acid can be modified at the 3′ nucleotide. For example, the two terminal hydroxyl groups of the 3′-most sugar can be oxidized to aldehyde groups and a concomitant opening of the ring to afford a modified nucleoside, analogous to the first ribonucleotide shown in the previous section entitled “Modified gRNAs”.

In another embodiment, the 3′ terminal sugar can be modified with a 2′3′ cyclic phosphate, analogous to the second ribonucleotide shown in the previous section entitled “Modified gRNAs”.

In some embodiments, the template nucleic acid may contain 3′ nucleotides which can be stabilized against degradation, e.g., by incorporating one or more of the modified nucleotides described herein. In this embodiment, e.g., thymines can be replaced with any of the modified thymines described herein; adenosines and guanosines can be replaced with modified adenosines and guanosines, e.g., with modifications at the 8-position, e.g., 8-bromo guanosine, or with any of the modified adenosines or guanosines described herein. In some embodiments, deaza nucleotides, e.g., 7-deaza-adenosine, can be incorporated into the template nucleic acid. In some embodiments, O- and N-alkylated nucleotides, e.g., N6-methyl andenosine, can be incorporated into the template nucleic acid. In some embodiments, sugar-modified deoxyribonucleotides can be incorporated, e.g., wherein the 2′ H-group is replaced by a group selected from OH, —OR, —R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), halo, —SH, —SR (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), amino (wherein amino can be, e.g., NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or cyano (—CN). In some embodiments, the phosphate backbone can be modified as described herein, e.g., with a phosphothioate group. In some embodiments, the template nucleic acid comprises an overhang region, and the nucleotides in the overhang region can each independently be a modified or unmodified nucleotide including, but not limited to 2′-sugar modified, such as, 2-F 2′-O-methyl, 2′-O-methoxyethyl-5-methyluridine (Teo), 2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinations thereof.

In some embodiments, the template nucleic acid is nicked, e.g., at the same position as a nick or DSB on target nucleic acid. While not wishing to be bound by theory, in some embodiments, a nick on a double stranded template nucleic acid stimulates HDR. In some embodiments, one or more nicks on the template nucleic acid are on the strand that is complementary to the intact target strand; in embodiments, one or more nicks on the template nucleic acid are on the strand that is complementary to the nicked target stand.

miRNA Binding Sites

MicroRNAs (or miRNAs) are naturally occurring cellular 19-25 nucleotide long noncoding RNAs. They bind to nucleic acid molecules having an appropriate miRNA binding site, e.g., in the 3′ UTR of an mRNA, and down-regulate gene expression. While not wishing to be bound by theory it is believed that the down regulation is either by reducing nucleic acid molecule stability or by inhibiting translation. An RNA species disclosed herein, e.g., an mRNA encoding Cas9 can comprise an miRNA binding site, e.g., in its 3′UTR. The miRNA binding site can be selected to promote down regulation of expression is a selected cell type. By way of example, the incorporation of a binding site for miR-122, a microRNA abundant in liver, can inhibit the expression of the gene of interest in the liver.

XI. Nucleic Acids; Kits; Methods of Production

In some aspects, disclosed herein is a nucleic acid, e.g., an isolated or non-naturally occurring nucleic acid, e.g., DNA, that comprises a sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a target domain as disclosed herein. In an embodiment, the nucleic acid encodes a gRNA molecule, e.g., a first gRNA molecule, comprising a targeting domain configured to provide a cleavage event, e.g., a double strand break or a single strand break, sufficiently close to a region desired to be altered to allow alteration, e.g., alteration associated with HDR of the region desired to be altered.

A nucleic acid disclosed herein may comprise (a) a sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a target domain as disclosed herein; (b) a sequence that encodes a Cas9 molecule; and further comprises (c) (i) a sequence that encodes a second gRNA molecule described herein having a targeting domain that is complementary to a second target domain, and optionally, (ii) a sequence that encodes a third gRNA molecule described herein having a targeting domain that is complementary to a third target domain; and optionally, (iii) a sequence that encodes a fourth gRNA molecule described herein having a targeting domain that is complementary to a fourth target domain.

In some embodiments, when a region that is desired to be altered is corrected by HDR (e.g., alt-HR, SSA, or HR), the nucleic acid encodes (a) a sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a target domain gene as disclosed herein; (b) a sequence that encodes a Cas9 molecule; (c) a template nucleic acid, (d) a HDR enhancer, and optionally, (e) (i) a sequence that encodes a second gRNA molecule described herein having a targeting domain that is complementary to a second target domain, and further optionally, (ii) a sequence that encodes a third gRNA molecule described herein having a targeting domain that is complementary to a third target domain; and still further optionally, (iii) a sequence that encodes a fourth gRNA molecule described herein having a targeting domain that is complementary to a fourth target domain.

As described above, a nucleic acid may comprise (a) a sequence encoding a gRNA molecule, and (b) a sequence encoding a Cas9 molecule. In some embodiments, (a) and (b) are present on the same nucleic acid molecule, e.g., the same vector, e.g., the same viral vector, e.g., the same adeno-associated virus (AAV) vector. In an embodiment, the nucleic acid molecule is an AAV vector. Exemplary AAV vectors that may be used in any of the described compositions and methods include an AAV2 vector, a modified AAV2 vector, an AAV3 vector, a modified AAV3 vector, an AAV6 vector, a modified AAV6 vector, an AAV8 vector and an AAV9 vector.

In other embodiments, (a) is present on a first nucleic acid molecule, e.g., a first vector, e.g., a first viral vector, e.g., a first AAV vector; and (b) is present on a second nucleic acid molecule, e.g., a second vector, e.g., a second vector, e.g., a second AAV vector. The first and second nucleic acid molecules may be AAV vectors.

In some embodiments, all of (a), (b), (c), (d), and optionally (e) are on the same vector, e.g., the same AAV vector. (It is understood that when the HDR enhancer (d) is on a nucleic acid or vector, it is implied that the HDR-enhancer molecule is capable of being encoded on a vector, e.g., the HDR-enhancer may be a protein, a ribozyme, a siRNA, an RNAi oligonucleotides, and the like.) In some embodiments, all of (a), (b), (c), (d), and optionally (e) are on different vectors, e.g., one or more AAV vectors. In some embodiments, two or more, e.g., two, three, or four of (a), (b), (c), (d), and optionally (e) are on the same vector, e.g., an AAV vector, and the remainder are on one or more other vectors, e.g., one or more AAV vectors. In some embodiments, (a) and (b) are on the same vector, e.g., an AAV vector; (a) and (c) are on the same vector, e.g., an AAV vector, (a) and (d) are on the same vector, e.g., an AAV vector, (b) and (c) are on the same vector, e.g., an AAV vector, (b) and (d) are on the same vector, e.g., an AAV vector, or (c) and (d) are on the same vector, e.g., an AAV vector.

The nucleic acids described herein may comprise a promoter operably linked to the sequence that encodes the gRNA molecule of (a), e.g., a promoter described herein. The nucleic acid may further comprise a second promoter operably linked to the sequence that encodes the second, third and/or fourth gRNA molecule of (e), e.g., a promoter described herein. The promoter and second promoter differ from one another. In some embodiments, the promoter and second promoter are the same.

The nucleic acids described herein may further comprise a promoter operably linked to the sequence that encodes the Cas9 molecule of (b), e.g., a promoter described herein.

The nucleic acids described herein may further comprise a promoter operably linked to the sequence that encodes the HDR-enhancer of (d), e.g., a promoter described herein.

In another aspect, disclosed herein is a kit comprising one or more, e.g., all of the following:

-   -   (a) gRNA molecule described herein, or nucleic acid that encodes         the gRNA;     -   (b) a Cas9 molecule, e.g., a Cas9 molecule described herein, or         a nucleic acid or mRNA that encodes the Cas9;     -   (c) a template nucleic acid; and     -   (d) an HDR-enhancer.

In an embodiment, the kit comprises nucleic acid, e.g., an AAV vector, that encodes one or more of (a), (b), (c), and (d).

XII. Methods of Treatment

A genetic disease is caused by a mutation in the patient's genome. Often, the mutation results in a change in a protein, e.g., an amino acid substitution or a truncation. Genetic diseases can be dominant, i.e., one mutant gene is sufficient to cause the disease, or recessive, where a patient with one copy of the mutant gene is an asymptomatic carrier, and two copies of the mutant gene are necessary for the disease to result.

Disclosed herein are the approaches to treat or prevent genetic diseases, using the compositions and methods described herein.

One approach to treat or prevent genetic diseases is to repair (i.e., correct) one or more mutations in the disease-causing gene by HDR. In this approach, mutant allele(s) are corrected and restored to wild type state. While not wishing to be bound by theory, it is believed that correction of the mutation to the corresponding wild-type sequence restores wild type protein production within the relevant cell type. The method described herein can be performed in all cell types.

In an embodiment, one mutant allele is repaired in the subject. For example, in a patient with an autosomal dominant genetic disease, the sole mutant allele in the cell is corrected so that the cell becomes wild-type at both loci. As another example, in a patient with an autosomal recessive genetic disease, one of the two mutant alleles in the cell is corrected, and so the cell becomes heterozygous, which is sufficient for normal functioning. As a recessive genetic disease only displays a phenotype when both alleles are mutated, repair of a single allele is adequate for a cure. In another embodiment, both mutant alleles are repaired in the subject. In either situation, the subjects can be cured of disease.

Correction of a mutation in the relevant gene may be performed prior to disease onset (e.g., prior to the appearance of symptoms) or after disease onset, for instance, early in the disease course.

In an embodiment, the method comprises initiating treatment of a subject prior to disease onset. In an embodiment, the method comprises initiating treatment of a subject after disease onset. In an embodiment, the method comprises initiating treatment of a subject well after disease onset, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, or 36 months after onset of the disease. While not wishing to be bound by theory it is believed that this may be effective if subjects did not present to physician until well into the course of illness.

In an embodiment, the method comprises initiating treatment of a subject in an advanced stage of disease.

Overall, initiation of treatment for subjects at all stages of disease is expected to prevent negative consequences of disease and be of benefit to subjects.

In an embodiment, the method comprises initiating treatment of a subject prior to disease expression. In an embodiment, the method comprises initiating treatment of a subject in an early stage of disease, e.g., when a subject has tested positive for the disease but has no signs or symptoms associated with the disease.

In an embodiment, the method comprises initiating treatment of a subject who has tested positive for the mutation underlying the disease, based on diagnosis via electrophoresis, genotyping, family history or other diagnostic criteria.

EXAMPLES

The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.

Introduction: Pathway Choice after DNA Double-Stranded Break Formation

After the formation of a DNA double-stranded break (DSB), the major decision point affecting DNA repair pathway choice is whether or not the DNA ends are endo- and exonucleolytically processed in a process referred to as end resection (FIG. 1 ). When no end resection takes places, the repair pathway engaged to repair the DSB is referred to as classical non-homologous end joining (C-NHEJ). The C-NHEJ repair pathway leads to either perfect repair of the DSBs, in which case the locus is restored without sequence alterations, or to the formation of small insertions and deletions.

In contrast, if the end resection machinery processes the DSB, a 3′ overhang is exposed, which engages in homology search. When the end resection is extensive, the exposed 3′ overhang can undergo strand invasion of highly homologous sequences, followed by repair of the DSB by a homology-dependent recombination (HDR) pathway. The HDR pathway comprises homologous recombination (HR), single strand annealing (SSA), and a potential third, not yet fully characterized alternative-HR pathway (“alt-HR”).

Yet another not completely characterized class of pathways that can engage the repair of DSBs after resection is initiated is referred to as alternative non-homologous end joining (ALT-NHEJ). Examples of pathways that are categorized as ALT-NHEJ include blunt end-joining (blunt EJ) and microhomology mediated end joining (MMEJ) leading to deletions, as well as synthesis dependent micro homology mediated end joining (SD-MMEJ), leading to the formation of insertions.

Overview of Different Cas9 Variants and gRNA Combinations

The wild-type (wt) Cas9 enzyme is directed to double-stranded DNA with a gRNA complementary to the desired genomic location, with the only requirement being the presence of a protospacer-adjacent motif (PAM) 3′ of the DNA target site. A wt Cas9 molecule then introduces two single nicks on opposite strands through the coordinated catalytic activity of the HNH and RuvC catalytic domains, leading to the formation of a blunt DSB (middle panel of FIG. 2 ). The HNH domain cleaves the strand complementary to the gRNA, while the RuvC domain places a cleavage event on the non-complementary strand.

Mutations in the HNH or RuvC domains can lead to the inactivation of their respective catalytic activities, thereby preventing the placement of a cleavage event on their respective target DNA strand. For example, a mutation in the RuvC domain of Cas9 (for example the D10A mutation of S. pyogenes Cas9) leads to the placement of a cleavage event only on the gRNA complementary strand through the remaining HNH domain activity when a single gRNA is used (upper panel, middle of FIG. 2 )). In contrast, an inactivating mutation of the HNH domain (for example the N863A mutation of S. pyogenes Cas9) leads to the cleavage of the non-complementary strand by the RuvC domain when a single gRNA is used (lower panel, middle of FIG. 2 ). The Cas9 catalytic domain mutants can also be used in combination with two gRNAs, which direct the single cleavage events to two different locations. For example, two gRNAs targeting opposite strands, in which PAMs face outwards, lead to the formation of 5′ protruding arms with D10A Cas9 mutants (RuvC domain catalytic mutant), in which the HNH domains place cleavage events on the gRNA complementary strands (upper panel, right of FIG. 2 ). Similarly, two gRNAs that direct the N863A Cas9 nickase (HNH domain catalytic mutant) to two opposite DNA strands in an orientation in which the PAMs face outwards, lead to the formation of 3′ protruding arms induced by non-complementary strand cleavage by the RuvC domains (lower panel, right of FIG. 2 ).

Example 1: Cleavage by Different Cas9 Variants Leads to Different Repair Outcomes

The different repair outcomes induced in response to DSBs introduced by different Cas9 variants were examined (FIG. 3 ). The CRISPR/Cas9 system was used to target the human HBB gene in the region of the sickle cell anemia-causing mutation. Blunt DSBs and dual-nicks in which the nicks are placed on opposite strands and with PAMs facing outwards, leaving either 3′ or 5′ overhangs as described above and as shown in FIG. 2 , were introduced by utilizing the wild type Cas9 nuclease, as well as the N863A or D10A Cas9 nickase mutants. Several different DNA repair outcomes including, e.g., indel mutations resulting from non-homologous end-joining, alternative NHEJ, HDR homologous recombination (HR) using the closely related HBD gene as an endogenous template, or homology dependent repair using an exogenous donor sequence were characterized.

Specifically, U20S cells were electroporated with 200 ng of each gRNA (HBB-8 and HBB-15), 750 ng of plasmid that encodes wild type Cas9 or mutant Cas9 (D10A or N863A). For some conditions, 50 pmols of single stranded oligonucleotide donor (ss-ODN) was included. Cells were collected 6 days after electroporation and genomic DNA was extracted. PCR amplification of the HBB locus was performed and subcloned into a Topo Blunt Vector. For each condition in each experiment (more the 4 independent samples) 98 colonies were analyses were sequenced with Sanger sequencing and scored for either insertions, deletions, gene conversion or gene corrections. In the absence of an exogenous ss-ODN (FIG. 3 ), a blunt DSB induced by WT Cas9 leads to the formation of predominantly (˜3/4 of all editing events) small deletions, followed by gene conversion and insertions. This is consistent with the notion that wt Cas9 generates blunt ends which are preferentially repaired by c-NHEJ. In contrast, DSBs induced by the N863A nickase mutant that leads to the formation of 3′ overhangs with the gRNA configuration used, are predominantly repaired by insertions in which the inserted sequence was a duplication of the overhang sequence (FIG. 3 ). This repair signature is indicative of repair by the ALT-NHEJ pathway. Lastly, the DSBs harboring a 5′ overhang induced by the D10A nickase mutants are predominantly repaired by the gene conversion pathway, which us a subset of the resection dependent HDR pathway. While the overall modification distribution remains similar in the presence of an exogenous ss-ODN (right panel of FIG. 4 ), we found that the most efficient repair using the ss-ODN as a template occurs in the presence of D10A induced lesions.

In summary, Cas9 nickases (D10A and N863A) showed comparable levels of efficacy compared to wildtype Cas9. However, different DNA ends engage different repair pathways. Wildtype Cas9 generates blunt ends, which are preferentially repaired by canonical NHEJ. Use of a Cas9 nickase with two gRNAs generates either 3′ or 5′ overhangs, which are most likely not suitable substrates to be repaired by canonical NHEJ but can be repaired by alternative pathways.

The 5′ protruding end was mostly repaired through a mechanism called gene conversion in which the HBB gene is repaired by using the HBD locus as a template. In addition, the D10A Nickase leaving a 5′ protruding arm is advantageous to promote HDR mediated by SS-ODN.

Example 2: WT Cas9-Induced Blunt DSBs are Repaired Through the C-NHEJ Pathway

In this experiment, the deletions occurring in response to different end structures induced by different Cas9 variants were analyzed. Initially, U2OS cells were electroporated with 200 ng of each gRNA (8 and 15), 750 ng of plasmid that encodes wild type Cas9 or mutant Cas9 (D10A or N863A). Cells were collected 6 days after electroporation and genomic DNA was extracted. PCR amplification of the HBB locus was performed and subcloned into a Topo Blunt Vector. For each condition in each experiment (more the 4 independent samples), 98 colonies were analyses were sequenced with Sanger sequencing. Deletions detected by Sanger sequencing were scored for the exact number of nucleotides deleted. The resulting deletion length data was represented on a histogram plot (FIG. 5 ).

Deletions occurring in response to blunt DSBs induced by the wt Cas9 molecule are predominantly small in size (green bars), which is indicative of repair through the c-NHEJ pathway. In contrast, the deletions observed in response to the 5′ and 3′ protruding DSBs induced by the D10A and N863A Cas9 variants were mostly larger in nature, indicating the activity of a different repair process.

Example 3: Modulation of Repair Pathway Choice by Inhibition of C-NHEJ Factors in the Context of a Wild-Type Cas9

Blunt DSBs get most frequently repaired by C-NHEJ, resulting in small deletions. C-NHEJ is a pathway that repairs DSBs that have not undergone extensive end processing by end resection. Many of the main components of the C-NHEJ pathway have been previously characterized (FIG. 6 ). The proteins 53BP1, Rif1, and PTIP are required for the initial pathway choice by preventing end resection from occurring. Proteins involved in the C-NHEJ repair process itself include the Ku70/Ku80 complex, DNA-PK, Artemis, Ligase 4, XRCC4 and XLF. Transient inhibition or down regulation of the Ku70/Ku80 complex, DNA-PK, Artemis, Ligase 4, XRCC4 and XLF is expected to yield to a shift in the balance from C-NHEJ towards alternative-NHEJ or HDR.

An alternative approach to enhance repair pathways other than C-NHEJ is to downregulate or transiently inhibit the proteins 53BP1, Rif1, and PtIP, which prevent end resection. It is expected that transient inhibition or down regulation of proteins that prevent end resection leads to the initiation of end resection and thereby influences pathway choice towards the resection dependent ALT-NHEJ and HDR pathways. Cells have also evolved pathways to protect against unwanted recombination. These anti-recombination factors include proteins such as Fbh1, RTEL, PARI and Rap80, as well as micro RNAs such as miRNA155, 545, 107, 1255, 148 and 193. A transient inhibition or down regulation of these proteins or inhibition of miRNAs with their respective antisense miRNAs are expected to increase usage of recombination-dependent pathways such as HDR.

Example 4: Overexpression of a Dominant Negative Form of 53BP1 Will Enhance Repair of DSBs by Resection Dependent Pathways

U2OS cells are electroporated with 200 ng of each gRNA (8 and 15), 750 ng of plasmid that encodes wild type Cas9, and a plasmid that encodes a dominant negative form of 53BP1 (see sequences, below) or a non-coding control plasmid, as an example of the inhibition of c-NHEJ. Cells are collected 6 days after electroporation and genomic DNA is extracted. PCR amplification of the HBB locus is performed and is subcloned into a Topo Blunt Vector. For each condition in each experiment colonies are sequenced with Sanger sequencing and HDR levels are scored.

53BP1 dominant negative Mutant 1: 53bp1_(1221- 1718)-with ATG start and TAA stop AA-seq (SEQ ID NO: 2250) MGEEEFDMPQPPHGHVLHRHMRTIREVRTLVTRVITDVYYVDGTEVERKV TEETEEPIVECQECETEVSPSQTGGSSGDLGDISSFSSKASSLHRTSSGT SLSAMHSSGSSGKGAGPLRGKTSGTEPADFALPSSRGGPGKLSPRKGVSQ TGTPVCEEDGDAGLGIRQGGKAPVTPRGRGRRGRPPSRTTGTRETAVPGP LGIEDISPNLSPDDKSFSRVVPRVPDSTRRTDVGAGALRRSDSPEIPFQA AAGPSDGLDASSPGNSFVGLRVVAKWSSNGYFYSGKITRDVGAGKYKLLF DDGYECDVLGKDILLCDPIPLDTEVTALSEDEYFSAGVVKGHRKESGELY YSIEKEGQRKWYKRMAVILSLEQGNRLREQYGLGPYEAVTPLTKAADISL DNLVEGKRKRRSNVSSPATPTASSSSSTTPTRKITESPRASMGVLSGKRK LITSEEERSPAKRGRKSATVKPGAVGAGEFVSPCESGDNTGEPSALEEQ* Nucleotide Seq (SEQ ID NO: 2251) ATGGGAGAAGAAGAGTTTGATATGCCTCAGCCTCCACATGGCCATGTCTT ACATCGTCACATGAGAACAATCCGGGAAGTACGCACACTTGTCACTCGTG TCATTACAGATGTGTATTATGTGGATGGAACAGAAGTAGAAAGAAAAGTA ACTGAGGAGACTGAAGAGCCAATTGTAGAGTGTCAGGAGTGTGAAACTGA AGTTTCCCCTTCACAGACTGGGGGCTCCTCAGGTGACCTGGGGGATATCA GCTCCTTCTCCTCCAAGGCATCCAGCTTACACCGCACATCAAGTGGGACA AGTCTCTCAGCTATGCACAGCAGTGGAAGCTCAGGGAAAGGAGCCGGACC ACTCAGAGGGAAAACCAGCGGGACAGAACCCGCAGATTTTGCCTTACCCA GCTCCCGAGGAGGCCCAGGAAAACTGAGTCCTAGAAAAGGGGTCAGTCAG ACAGGGACGCCAGTGTGTGAGGAGGATGGTGATGCAGGCCTTGGCATCAG ACAGGGAGGGAAGGCTCCAGTCACGCCTCGTGGGCGTGGGCGAAGGGGCC GCCCACCTTCTCGGACCACTGGAACCAGAGAAACAGCTGTGCCTGGCCCC TTGGGCATAGAGGACATTTCACCTAACTTGTCACCAGATGATAAATCCTT CAGCCGTGTCGTGCCCCGAGTGCCAGACTCCACCAGACGAACAGATGTGG GTGCTGGTGCTTTGCGTCGTAGTGACTCTCCAGAAATTCCTTTCCAGGCT GCTGCTGGCCCTTCTGATGGCTTAGATGCCTCCTCTCCAGGAAATAGCTT TGTAGGGCTCCGTGTTGTAGCCAAGTGGTCATCCAATGGCTACTTTTACT CTGGGAAAATCACACGAGATGTCGGAGCTGGGAAGTATAAATTGCTCTTT GATGATGGGTACGAATGTGATGTGTTGGGCAAAGACATTCTGTTATGTGA CCCCATCCCGCTGGACACTGAAGTGACGGCCCTCTCGGAGGATGAGTATT TCAGTGCAGGAGTGGTGAAAGGACATAGGAAGGAGTCTGGGGAACTGTAC TACAGCATTGAAAAAGAAGGCCAAAGAAAGTGGTATAAGCGAATGGCTGT CATCCTGTCCTTGGAGCAAGGAAACAGACTGAGAGAGCAGTATGGGCTTG GCCCCTATGAAGCAGTAACACCTCTTACAAAGGCAGCAGATATCAGCTTA GACAATTTGGTGGAAGGGAAGCGGAAACGGCGCAGTAACGTCAGCTCCCC AGCCACCCCTACTGCCTCCAGTAGCAGCAGCACAACCCCTACCCGAAAGA TCACAGAAAGTCCTCGTGCCTCCATGGGAGTTCTCTCAGGCAAAAGAAAA CTTATCACTTCTGAAGAGGAACGGTCCCCTGCCAAGCGAGGTCGCAAGTC TGCCACAGTAAAACCTGGTGCAGTAGGGGCAGGAGAGTTTGTGAGCCCCT GTGAGAGTGGAGACAACACCGGTGAACCCTCTGCCCTGGAAGAGCAGTAA 53BP1 dominant negative Mutant 2: 53bp1_(1052- 1710)-with ATG start and TAA stop AA-seq (SEQ ID NO: 2252) MDPPTTPIRGNLLHFPSSQGEEEKEKLEGDHTIRQSQQPMKPISPVKDPV SPASQKMVIQGPSSPQGEAMVTDVLEDQKEGRSTNKENPSKALIERPSQN NIGIQTMECSLRVPETVSAATQTIKNVCEQGTSTVDQNFGKQDATVQTER GSGEKPVSAPGDDTESLHQGEEEFDMPQPPHGHVLHRHMRTIREVRTLVT RVITDVYYVDGTEVERKVTEETEEPIVECQECETEVSPSQTGGSSGDLGD ISSFSSKASSLHRTSSGTSLSAMHSSGSSGKGAGPLRGKTSGTEPADFAL PSSRGGPGKLSPRKGVSQTGTPVCEEDGDAGLGIRQGGKAPVTPRGAAAG PSDGLDASSPGNSFVGLRVVAKWSSNGYFYSGKITRDVGAGKYKLLFDDG YECDVLGKDILLCDPIPLDTEVTALSEDEYFSAGVVKGHRKESGELYYSI EKEGQRKWYKRMAVILSLEQGNRLREQYGLGPYEAVTPLTKAADISLDNL VEGKRKRRSNVSSPATPTASSSSSTTPTRKITESPRASMGVLSGKRKLIT SEEERSPAKRGRKSATVKPGAVGAGEFVSPCESGDNTG* Nucleotide Seq (SEQ ID NO: 2253) ATGGATCCCCCCACCACACCCATCAGGGGGAACTTGCTCCACTTTCCAAG TTCTCAAGGAGAAGAGAAAGAAAAATTGGAGGGTGACCATACAATCAGGC AGAGTCAACAGCCTATGAAGCCCATTAGTCCTGTCAAGGACCCTGTTTCT CCTGCTTCCCAGAAGATGGTCATACAAGGGCCATCCAGTCCTCAAGGAGA GGCAATGGTGACAGATGTGCTAGAAGACCAGAAAGAAGGACGGAGTACTA ATAAGGAAAATCCTAGTAAGGCCTTGATTGAAAGGCCCAGCCAAAATAAC ATAGGAATCCAAACCATGGAGTGTTCCTTGAGGGTCCCAGAAACTGTTTC AGCAGCAACCCAGACTATAAAGAATGTGTGTGAGCAGGGGACCAGTACAG TGGACCAGAACTTTGGAAAGCAAGATGCCACAGTTCAGACTGAGAGGGGG AGTGGTGAGAAACCAGTCAGTGCTCCTGGGGATGATACAGAGTCGCTCCA TAGCCAGGGAGAAGAAGAGTTTGATATGCCTCAGCCTCCACATGGCCATG TCTTACATCGTCACATGAGAACAATCCGGGAAGTACGCACACTTGTCACT CGTGTCATTACAGATGTGTATTATGTGGATGGAACAGAAGTAGAAAGAAA AGTAACTGAGGAGACTGAAGAGCCAATTGTAGAGTGTCAGGAGTGTGAAA CTGAAGTTTCCCCTTCACAGACTGGGGGCTCCTCAGGTGACCTGGGGGAT ATCAGCTCCTTCTCCTCCAAGGCATCCAGCTTACACCGCACATCAAGTGG GACAAGTCTCTCAGCTATGCACAGCAGTGGAAGCTCAGGGAAAGGAGCCG GACCACTCAGAGGGAAAACCAGCGGGACAGAACCCGCAGATTTTGCCTTA CCCAGCTCCCGAGGAGGCCCAGGAAAACTGAGTCCTAGAAAAGGGGTCAG TCAGACAGGGACGCCAGTGTGTAGGAGGATGGTGATGCAGGCCTTGGCAT CAGACAGGGAGGGAAGGCTCCAGTCACGCCTCGTGGGCGTGGGCGAAGGG GCCGCCCACCTTCTCGGACCACTGGAACCAGAGAAACAGCTGTGCCTGGC CCCTTGGGCATAGAGGACATTTCACCTAACTTGTCACCAGATGATAAATC CTTCAGCCGTGTCGTGCCCCGAGTGCCAGACTCCACCAGACGAACAGATG TGGGTGCTGGTGCTTTGCGTCGTAGTGACTCTCCAGAAATTCCTTTCCAG GCTGCTGCTGGCCCTTCTGATGGCTTAGATGCCTCCTCTCCAGGAAATAG CTTTGTAGGGCTCCGTGTTGTAGCCAAGTGGTCATCCAATGGCTACTTTT ACTCTGGGAAAATCACACGAGATGTCGGAGCTGGGAAGTATAAATTGCTC TTTGATGATGGGTACGAATGTGATGTGTTGGGCAAAGACATTCTGTTATG TGACCCCATCCCGCTGGACACTGAAGTGACGGCCCTCTCGGAGGATGAGT ATTTCAGTGCAGGAGTGGTGAAAGGACATAGGAAGGAGTCTGGGGAACTG TACTACAGCATTGAAAAAGAAGGCCAAAGAAAGTGGTATAAGCGAATGGC TGTGATCCTGTCCTTGGAGCAAGGAAACAGACTGAGAGAGCAGTATGGGC TTGGCCCCTATGAAGCAGTAACACCTCTTACAAAGGCAGCAGATATCAGC TTAGACAATTTGGTGGAAGGGAAGCGGAAACGGCGCAGTAACGTCAGCTC CCCAGCCACCCCTACTGCCTCCAGTAGCAGCAGCACAACCCCTACCCGAA AGATCACAGAAAGTCCTCGTGCCTCCATGGGAGTTCTCTCAGGCAAAAGA AAACTTATCACTTCTGAAGAGGAACGGTCCCCTGCCAAGCGAGGTCGCAA GTCTGCCACAGTAAAACCTGGTGCAGTAGGGGCAGGAGAGTTTGTGAGCC CCTGTGAGAGTGGAGACAACACCGGTTAA

Example 5: Down Regulation of Artemis Expression Leads to Enhanced Gene Correction Efficiency by HDR

Artemis is a protein involved in the c-NHEJ repair pathway. To examine whether Artemis plays a role in the repair of CRISPR/Cas9 induced blunt DSBs, Artemis was downregulated using a CRISPR/Cas9 mediated knockout approach (FIG. 7 ). Specifically, U2OS cells were electroporated with 19 pmols of S. aureus Cas9 complexed with a gRNA directing the Cas9 protein to the Artemis locus. A second set of cells were electroporated with S. aureus Cas9 complexed with non-targeting control gRNA. Samples were collected on day 4 for Western Blot to assess the level of down regulation. On day 7, cells with down regulated Artemis expression and control cells were electroporated with 200 ng of gRNA8, 750 ng of plasmid that encodes wild type Cas9, and 50 pmols of a ss-ODN. Cells were collected 5 days after electroporation and genomic DNA was extracted. PCR amplification of the HBB locus was performed and the resulting products were subcloned into a Topo Blunt Vector. For each condition in each experiment, colonies were sequenced with Sanger sequencing. Upon down regulation of Artemis, an increase in HDR-mediated gene correction efficiency was observed (FIG. 7 ).

Example 6: Down Regulation of Anti-Recombination Factor Rap80 to Enhance DNA Repair by HDR

U2OS cells were electroporated with 200 ng of each gRNA (8 and 15), 750 ng of plasmid that encodes wild type and 30 pmols of an siRNA targeting the Rap80 transcript as 40 an example of down regulation of anti recombination factors or a control siRNA. Cells were collected for a Western Blot assay to check Rap80 protein levels 4 days after electroporation (FIG. 8 ), and genomic DNA is extracted. PCR amplification of the HBB locus is performed and is subcloned into a Topo Blunt Vector. For each condition in each experiment, colonies are sequenced with Sanger sequencing and HDR levels are scored

Example 7: Modulating Pathway Choice of Cas9 Induced DSBs with 3′ Overhangs

N863A N863A Cas9 nickase induced DSBs that lead to the formation of a 3′ overhang structure are predominantly repaired using insertions, which is indicative of the ALT-NHEJ pathway (FIG. 9 ). The ALT-NHEJ pathway comprises of several sub-pathways referred to as blunt end joining (B-EJ), microhomology mediated end joining (MMEJ) and synthesis dependent microhomology mediated end joining (SD-MMEJ). While MMEJ and B-EJ repair outcomes are mostly deletions, insertions are a result of the SD-MMEJ pathway. DNA repair pathway choice can be altered by transiently inhibiting or down regulating components of the ALT-NHEJ pathway, such as XRCC1, Ligase I, Ligase III and Polymerase theta. Upon down regulation or transient inhibition of these ALT-NHEJ components we expect to observe increases in DNA repair by either C-NHEJ or HDR mediated pathways.

Example 8: Down Regulation of Alt-NHEJ Factors to Enhance DNA Repair by HDR

U2OS cells were electroporated with 200 ng of each gRNA (8 and 15), 750 ng of plasmid that encodes N863A Cas9, and 30 pmols of an siRNA targeting the Pol Theta transcript as an example of inhibition of the alt-NHEJ or a control siRNA. Five days after electroporation genomic DNA was extracted. PCR amplification of the HBB locus was performed and products were subcloned into a Topo Blunt Vector. For each condition in each experiment colonies were sequenced with Sanger sequencing.

As shown in FIG. 10 , down regulation of Pol theta in the context of the N863A Cas9 nuclease leads to a strong reduction of the insertion frequency and an increase in the gene conversion rate. These data suggest that the 3′ protruding ends generated by the N863A Cas9 nuclease are substrates for processing by Pol Theta, resulting in a high accumulation of insertions. Upon down-regulation of Pol Theta, the 3′ protruding DNA ends are available for engaging the gene conversion pathway (FIG. 9 ).

Example 9: Modulating Pathway Choice of Cas9 Induced DSBs with 5′ Overhangs in the Presence of a Ss-ODN

The D10A Cas9 nickase induced DSBs that lead to the formation of 5′ overhangs most efficiently incorporate an exogenous ss-ODN donor template among the other Cas9 variants. The factors involved in the repair pathway through which the ss-ODN template is incorporated is incompletely characterized, but falls under the resection dependent homology-dependent (HDR) repair pathway, which includes the SSA, HR, and a potential third, alternative-HR pathway (alt-HR), for which no genetic requirements are yet identified. Factors involved in the HR pathway include Brca1, CtiP, Exo1, Brca2 and Rad51 (FIG. 11 ). By transiently inhibiting or down regulating factors involved in HR, an increase in SSA repair events should be observed, if HR and SSA are indeed competitive pathways. Also, a decrease in gene conversion upon down regulation or inhibition of HR factors is expected if gene conversion is in fact dependent in HR. If the ss-ODN incorporation is mediated through the SSA annealing pathway, an increase in in ss-ODN incorporation upon the transient inhibition or down regulation of HR components is expected, if HR and SSA are indeed competitive pathways.

Proteins involved in the SSA pathway include Rad52, ERCC1, XPF (SSA), and Ligase 1 (FIG. 11 ). By transiently inhibiting or down regulating factors involved in SSA, an increase in HR repair events is expected, if HR and SSA are indeed competitive pathways. If the ss-ODN incorporation is dependent on the SSA pathway, a decrease of donor integration upon transient inhibition or down regulation of the SSA pathway is expected. In contrast, if the donor integration is dependent on components of the HR pathway, and if SSA is indeed competing with the HR pathway, we expect to observe an increase in ss-ODN integration upon SSA pathway down regulation or inhibition.

Example 10: Down Regulation of HR Factors to Address the Mechanism of Gene Conversion Repair

Whether gene conversion in the context of the D10A Cas9 with 2 gRNAs is dependent on the HR pathway was evaluated (FIG. 12 ). U2OS cell were electroporated with 200 ng of each gRNA (8 and 15), 750 ng of plasmid that encodes D10A Cas9 and 30 pmol of scrambled siRNA as a negative control or 30 pmol of siRNA against BRCA2 or Rad51—central players of canonical HR—as an example, with or without 50 pmols of a ss-ODN. Five days after electroporation genomic DNA was extracted. PCR amplification of the HBB locus was performed and products were subcloned into a Topo Blunt Vector. For each condition in each experiment colonies were sequenced with Sanger sequencing.

As shown in FIG. 12 , the majority of the gene conversion events were mediated by BRCA2 and Rad51. These data are consistent with the notion that gene conversion using the HBD gene as a donor in cis is an event mediated by canonical HR. Rad51 and BRCA2 do not seams to effect the repair mediated by ss-ODN.

Example 11: Down Regulation of SSA Factors to Address the Mechanism of Gene Correction Repair

Next, whether the gene correction in the context of the D10A with 2 gRNA was dependent on the Single Strand Annealing (SSA) pathway was evaluated(FIG. 13 ). Specifically, U2OS cells were electroporated with 19 pmols of S. aureus Cas9 complexes with different gRNAs directing the Cas9 protein to the ERCC1 and Rad52 locus, as an example of SSA components. A second set of cells were electroporated with S. aureus Cas9 complexed with a non-targeting control gRNAs. Samples were collected on day 4 for Western Blot to assess the level of down regulation (FIG. 13 , panel B). On day 7, cells with down regulated ERCC1 or Rad52 expression and control cells were electroporated with 200 ng of gRNA8, 750 ng of plasmid that encodes D10A Cas9, with or without 50 pmols of a ss-ODN. Cells were collected 5 days after electroporation, and genomic DNA was extracted. PCR amplification of the HBB locus was performed and the resulting products were subcloned into a Topo Blunt Vector. For each condition in each experiment, colonies were sequenced with Sanger. A significant reduction in gene correction upon down regulation of the SSA pathway was observed, but not a complete elimination, suggesting that either the cells were not fully knocked down or indicative indeed of a presence of a third, alt-HR pathway.

Example 12: Down Regulation of HR Factors to Address the Mechanism of Gene Conversion Repair

Whether gene conversion in the context of the D10A Cas9 with 2 gRNAs is dependent on the canonical HR pathway was evaluated (FIG. 11 ). U2OS cell were electroporated with 200 ng of each gRNA (HBB-8 and HBB-15), 750 ng of plasmid that encodes D10A Cas9 and 30 pmol of scrambled siRNA as a negative control or 30 pmol of siRNA against Exo1—a key end resection factor required for exposing ssDNA overhangs—as an example, with or without 50 pmols of a ss-ODN. Cells were collected for Western Blot analysis on day 4. Five days after electroporation genomic DNA was extracted. PCR amplification of the HBB locus was performed and products were subcloned into a Topo Blunt Vector. For each condition in each experiment colonies were sequenced with Sanger sequencing.

As shown in the left panel of FIG. 14 , gene conversion was unexpectedly increased in the absence of Exo1. To confirm these unexpected results, a CRISPR/Cas9 mediated knockout approach against Exo1 was utilized. Specifically, U2OS cells were electroporated with 19 pmols of S. aureus Cas9 complexes with a gRNAs directing the Cas9 protein to the Exo1 locus. A second set of cells were electroporated with S. aureus Cas9 complexed with a non-targeting control gRNAs. Samples were collected on day 4 for Western Blot to assess the level of down regulation (FIG. 14 , right panel). On day 7, cells with down regulated Exo1 expression and control cells were electroporated with 200 ng of gRNA8, 750 ng of plasmid that encodes D10A Cas9. Cells were collected 5 days after electroporation and genomic DNA was extracted. PCR amplification of the HBB locus was performed and the resulting products were subcloned into a Topo Blunt Vector. For each condition in each experiment, colonies were sequenced with Sanger. Again, an increase in gene conversion frequency upon down regulation of Exo1 with the CRISPR/Cas9 system was observed.

Example 13: Modulating Pathway Choice of Cas9-Induced DSBs by Modulation of Epigenetic Modifiers

DSB repair is characterized by a its surrounding chromatin structure. A blunt DSB that is most frequently repaired by the C-NHEJ pathway is associated with a distinct signature of chromatin modifications that promote the pathway choice decision for a cell to undergo C-NHEJ. By modulating the factors responsible for placing or removing chromatin marks that impact on the pathway choice decision towards C-NHEJ, the repair balance should shift towards resection dependent pathways such as ALT-NHEJ or HDR. For example, proteins that affect DNA repair pathway choice include KDM4A, Setd2, HDAC1, HDAC2, and EZH2. By modulating the activity of these factors the balance of repair pathway choice should be altered.

Example 14: HDAC1 Inhibitor Treatment

U2OS cells were electroporated with 200 ng of each gRNA (HBB-8 and HBB-15), 750 ng of plasmid that encodes wild type, D10A, or N863A Cas9, and an inhibitor against HDAC1 (Trichostatin A (TSA) or Sodium Butyrate (NaB), for example). Cells are collected 6 days after electroporation and genomic DNA is extracted. PCR amplification of the HBB locus is performed and is subcloned into a Topo Blunt Vector. For each condition in each experiment colonies are sequenced with Sanger sequencing and HDR levels are scored.

Example 15: Table of Reagents

The below table lists siRNAs that may be useful in the described compositions and methods.

Gene Catalog Item BRCA2 M-003462-01-0005 siGENOME Human BRCA2 (675) siRNA - SMARTpool, 5 nmol Exo1 L-013120-00-0005 ON-TARGETplus Human EXO1 (9156) siRNA - SMARTpool, 5 nmol Rap80 L-006995-00-0005 ON-TARGETplus Human UIMC1- Rap80 (51720) siRNA - SMARTpool, 5 nmol Rap80 M-006995-03-0005 siGENOME Human UIMC1 (51720) siRNA - SMARTpool, 5 nmol Rad51 D-003530-05-0005 siGENOME Human RAD51 (5888) siRNA - Individual, 5 nmol Rad51 D-003530-07-0005 siGENOME Human RAD51 (5888) siRNA - Individual, 5 nmol FF CGUACGCGGAAUACUUCGAUU FF: siRNA, Standard 0.015 μmol (SEQ ID NO: 2254) Regular PolQ SI00090062 Hs_POLQ_1 FlexiTube siRNA, NM_006596, NM_199420

The below table lists some exemplary gRNAs for use in the described compositions and methods.

Gene ID Gene name Target 10721 POLQ_1_WS147 GGAGTGGGAAACGGCGGCGTT 10721 POLQ_2_WS148 GTGCCAGCCCCCAGTTCCTCT 10721 POLQ_3_WS149 GTGTCACCGCCGCTTCCCGAGA 5893 Rad52_gRNA#3 (Exon1) AGGATGGCTGTCACGTCCTCC 5893 Rad52_gRNA#4 (Exon1) ACACTGAGCCGCCGCCAGCAG 5893 Rad52_gRNA#5 (Exon 1) TGTGGTTCCTTCATGCCGGGA 5893 Rad52_gRNA#6 (Exon 1) GCCGGGAAAGAATGAGGTGAA 5893 Rad52_gRNA#7 (Exon2) ACATAAGTAGCCGCATGGCTG 5893 Rad52_gRNA#8 (Exon2) CCCTGAGGCAGAGGCTGGGCC 5893 Rad52_gRNA#9 (Exon2) CAGCCTCTGCCTCAGGGCCTT 5893 Rad52_gRNA#10 (Exon2) TGCAGTGCCAGTACACAGCAG 9156 Exo1_gRNA#3 AATTTGGCACCATGGGGATAC 9156 Exo1_gRNA#4 CCTTTATACTTCCTCACATGG 64421 DCLRE1C_gRNA4 CGAAGCGGTCTATGGAGATAG 64421 DCLRE1C_gRNA5 TCCTGTCCCACTGCCACAAAG 4361 Mre11A_gRNA#4 TAGTTGCAACAGATATTCATC 4361 Mre11A_gRNA#5 TGGGCAAGTCTTAAAATTTCA 4361 Mre11A_gRNA#6 ATCTTTCTCCATAAATCCAAG 2067 ERCC1_gRNA_1 GGAGGGACCTCATCCTCGTCG 2067 ERCC1_gRNA_2 GCACCCCCTCTTTGTCCTTCC 2067 ERCC1_gRNA_3 AAGAGGGGGTGCCCCAGCCCT 2067 ERCC1_gRNA_4 AAATTTGTGATACCCCTCGAC 2067 ERCC1_gRNA_5 CGACGAGGATGAGGTCCCTCC 5932 RBBP8_1_WS143 GTAAAGTCACTAGATGTATCTG 5932 RBBP8_2_WS144 GAGGACCTTTGGACAAAACTAA Amino acid sequences described herein:

SEQ ID NO: 6 MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSEL SGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRG SINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKY AYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDI KDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQI AIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRN RQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENS KKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYF RVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETE QEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMY HHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYR FDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNM IDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG SEQ ID NO: 7 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQ EIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRG HFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLT PNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDL TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIH LGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNF DKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEIS GVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSR KLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDI NRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTA LIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRD FATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKE LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKL KGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFD TTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SEQ ID NO: 8 MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLALPRRLARSARKRLARRKARLNHLKHLIANEFKLNYEDY QSFDESLAKAYKGSLISPYELRFRALNELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVGEYL YKEYFQKFKENSKEFTNVRNKKESYERCIAQSFLKDELKLIFKKQREFGFSFSKKFEEEVLSVAFYKRALKDFSHLVGNCSFF TDEKRAPKNSPLAFMFVALTRIINLLNNLKNTEGILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYEFKGEKGTYFIEF KKYKEFIKALGEHNLSQDDLNEIAKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFKALKLVTPLMLEGKKYD EACNELNLKVAINEDKKDFLPAFNETYYKDEVTNPVVLRAIKEYRKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQ NENYKAKKDAELECEKLGLKINSKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVLVFTKQ NQEKLNQTPFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKDRNLNDTRYIARLVLNYTKDYLDFLPLSDDE NTKLNDTQKGSKVHVEAKSGMLTSALRHTWGFSAKDRNNHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISEL DYKNKRKFFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKEGVLKALELGKIRKVNGKIVKNGDM FRVDIFKHKKTNKFYAVPIYTMDFALKVLPNKAVARSKKGEIKDWILMDENYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFT SSTVSLIVSKHDNKFETLSKNQKILFKNANEKEVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQREDFKK SEQ ID NO: 9 MKRILGLDLGTNSIGWALVNEAENKDERSSIVKLGVRVNPLTVDELTNFEKGKSITTNADRTLKRGMRRNLQRYKLRRETLTE VLKEHKLITEDTILSENGNRTTFETYRLRAKAVTEEISLEEFARVLLMINKKRGYKSSRKAKGVEEGTLIDGMDIARELYNNN LTPGELCLQLLDAGKKFLPDFYRSDLQNELDRIWEKQKEYYPEILTDVLKEELRGKKRDAVWAICAKYFVWKENYTEWNKEKG KTEQQEREHKLEGIYSKRKRDEAKRENLQWRVNGLKEKLSLEQLVIVFQEMNTQINNSSGYLGAISDRSKELYFNKQTVGQYQ MEMLDKNPNASLRNMVFYRQDYLDEFNMLWEKQAVYHKELTEELKKEIRDIIIFYQRRLKSQKGLIGFCEFESRQIEVDIDGK KKIKTVGNRVISRSSPLFQEFKIWQILNNIEVIVVGKKRKRRKLKENYSALFEELNDAEQLELNGSRRLCQEEKELLAQELFI RDKMTKSEVLKLLFDNPQELDLNFKTIDGNKTGYALFQAYSKMIEMSGHEPVDFKKPVEKVVEYIKAVFDLLNWNTDILGFNS NEELDNQPYYKLWHLLYSFEGDNTPTGNGRLIQKMTELYGFEKEYATILANVSFQDDYGSLSAKAIHKILPHLKEGNRYDVAC VYAGYRHSESSLTREEIANKVLKDRLMLLPKNSLHNPVVEKILNQMVNVINVIIDIYGKPDEIRVELARELKKNAKEREELTK SIAQTTKAHEEYKTLLQTEFGLTNVSRTDILRYKLYKELESCGYKTLYSNTYISREKLFSKEFDIEHIIPQARLFDDSFSNKT LEARSVNIEKGNKTAYDFVKEKFGESGADNSLEHYLNNIEDLFKSGKISKTKYNKLKMAEQDIPDGFIERDLRNTQYIAKKAL SMLNEISHRVVATSGSVTDKLREDWQLIDVMKELNWEKYKALGLVEYFEDRDGRQIGRIKDWTKRNDHRHHAMDALTVAFTKD VFIQYFNNKNASLDPNANEHAIKNKYFQNGRAIAPMPLREFRAEAKKHLENTLISIKAKNKVITGNINKTRKKGGVNKNMQQT PRGQLHLETIYGSGKQYLTKEEKVNASFDMRKIGTVSKSAYRDALLKRLYENDNDPKKAFAGKNSLDKQPIWLDKEQMRKVPE KVKIVTLEAIYTIRKEISPDLKVDKVIDVGVRKILIDRLNEYGNDAKKAFSNLDKNPIWLNKEKGISIKRVTISGISNAQSLH VKKDKDGKPILDENGRNIPVDFVNTGNNHHVAVYYRPVIDKRGQLVVDEAGNPKYELEEVVVSFFEAVTRANLGLPIIDKDYK TTEGWQFLFSMKQNEYFVFPNEKTGFNPKEIDLLDVENYGLISPNLFRVQKFSLKNYVFRHHLETTIKDTSSILRGITWIDFR SSKGLDTIVKVRVNHIGQIVSVGEY SEQ ID NO: 10 MSRKNYVDDYAISLDIGNASVGWSAFTPNYRLVRAKGHELIGVRLFDPADTAESRRMARTTRRRYSRRRWRLRLLDALFDQAL SEIDPSFLARRKYSWVHPDDENNADCWYGSVLFDSNEQDKRFYEKYPTIYHLRKALMEDDSQHDIREIYLAIHHMVKYRGNFL VEGTLESSNAFKEDELLKLLGRITRYEMSEGEQNSDIEQDDENKLVAPANGQLADALCATRGSRSMRVDNALEALSAVNDLSR EQRAIVKAIFAGLEGNKLDLAKIFVSKEFSSENKKILGIYFNKSDYEEKCVQIVDSGLLDDEEREFLDRMQGQYNAIALKQLL GRSTSVSDSKCASYDAHRANWNLIKLQLRTKENEKDINENYGILVGWKIDSGQRKSVRGESAYENMRKKANVFFKKMIETSDL SETDKNRLIHDIEEDKLFPIQRDSDNGVIPHQLHQNELKQIIKKQGKYYPFLLDAFEKDGKQINKIEGLLTFRVPYFVGPLVV PEDLQKSDNSENHWMVRKKKGEITPWNFDEMVDKDASGRKFIERLVGTDSYLLGEPTLPKNSLLYQEYEVLNELNNVRLSVRT GNHWNDKRRMRLGREEKTLLCQRLFMKGQTVTKRTAENLLRKEYGRTYELSGLSDESKFTSSLSTYGKMCRIFGEKYVNEHRD LMEKIVELQTVFEDKETLLHQLRQLEGISEADCALLVNTHYTGWGRLSRKLLTTKAGECKISDDFAPRKHSIIEIMRAEDRNL MEIITDKQLGFSDWIEQENLGAENGSSLMEVVDDLRVSPKVKRGIIQSIRLIDDISKAVGKRPSRIFLELADDIQPSGRTISR KSRLQDLYRNANLGKEFKGIADELNACSDKDLQDDRLFLYYTQLGKDMYTGEELDLDRLSSAYDIDHIIPQAVTQNDSIDNRV LVARAENARKTDSFTYMPQIADRMRNFWQILLDNGLISRVKFERLTRQNEFSEREKERFVQRSLVETRQIMKNVATLMRQRYG NSAAVIGLNAELTKEMHRYLGFSHKNRDINDYHHAQDALCVGIAGQFAANRGFFADGEVSDGAQNSYNQYLRDYLRGYREKLS AEDRKQGRAFGFIVGSMRSQDEQKRVNPRTGEVVWSEEDKDYLRKVMNYRKMLVTQKVGDDFGALYDETRYAATDPKGIKGIP FDGAKQDTSLYGGFSSAKPAYAVLIESKGKTRLVNVTMQEYSLLGDRPSDDELRKVLAKKKSEYAKANILLRHVPKMQLIRYG GGLMVIKSAGELNNAQQLWLPYEEYCYFDDLSQGKGSLEKDDLKKLLDSILGSVQCLYPWHRFTEEELADLHVAFDKLPEDEK KNVITGIVSALHADAKTANLSIVGMTGSWRRMNNKSGYTFSDEDEFIFQSPSGLFEKRVTVGELKRKAKKEVNSKYRTNEKRL PTLSGASQP SEQ ID NO: 11 METQTSNQLITSHLKDYPKQDYFVGLDIGTNSVGWAVTNTSYELLKFHSHKMWGSRLFEEGESAVTRRGFRSMRRRLERRKLR LKLLEELFADAMAQVDSTFFIRLHESKYHYEDKTTGHSSKHILFIDEDYTDQDYFTEYPTIYHLRKDLMENGTDDIRKLFLAV HHILKYRGNFLYEGATFNSNAFTFEDVLKQALVNITFNCFDTNSAISSISNILMESGKTKSDKAKAIERLVDTYTVFDEVNTP DKPQKEQVKEDKKTLKAFANLVLGLSANLIDLFGSVEDIDDDLKKLQIVGDTYDEKRDELAKVWGDEIHIIDDCKSVYDAIIL MSIKEPGLTISQSKVKAFDKHKEDLVILKSLLKLDRNVYNEMFKSDKKGLHNYVHYIKQGRTEETSCSREDFYKYTKKIVEGL ADSKDKEYILNEIELQTLLPLQRIKDNGVIPYQLHLEELKVILDKCGPKFPFLHTVSDGFSVTEKLIKMLEFRIPYYVGPLNT HHNIDNGGFSWAVRKQAGRVTPWNFEEKIDREKSAAAFIKNLTNKCTYLFGEDVLPKSSLLYSEFMLLNELNNVRIDGKALAQ GVKQHLIDSIFKQDHKKMTKNRIELFLKDNNYITKKHKPEITGLDGEIKNDLTSYRDMVRILGNNFDVSMAEDIITDITIFGE SKKMLRQTLRNKFGSQLNDETIKKLSKLRYRDWGRLSKKLLKGIDGCDKAGNGAPKTIIELMRNDSYNLMEILGDKFSFMECI EEENAKLAQGQVVNPHDIIDELALSPAVKRAVWQALRIVDEVAHIKKALPSRIFVEVARTNKSEKKKKDSRQKRLSDLYSAIK KDDVLQSGLQDKEFGALKSGLANYDDAALRSKKLYLYYTQMGRCAYTGNIIDLNQLNTDNYDIDHIYPRSLTKDDSFDNLVLC ERTANAKKSDIYPIDNRIQTKQKPFWAFLKHQGLISERKYERLTRIAPLTADDLSGFIARQLVETNQSVKATTTLLRRLYPDI DVVFVKAENVSDFRHNNNFIKVRSLNHHHHAKDAYLNIVVGNVYHEKFTRNFRLFFKKNGANRTYNLAKMFNYDVICTNAQDG KAWDVKTSMNTVKKMMASNDVRVTRRLLEQSGALADATIYKASVAAKAKDGAYIGMKTKYSVFADVTKYGGMTKIKNAYSIIV QYTGKKGEEIKEIVPLPIYLINRNATDIELIDYVKSVIPKAKDISIKYRKLCINQLVKVNGFYYYLGGKTNDKIYIDNAIELV VPHDIATYIKLLDKYDLLRKENKTLKASSITTSIYNINTSTVVSLNKVGIDVFDYFMSKLRTPLYMKMKGNKVDELSSTGRSK FIKMTLEEQSIYLLEVLNLLTNSKTTFDVKPLGITGSRSTIGVKIHNLDEFKIINESITGLYSNEVTIV SEQ ID NO: 12 MTKLNQPYGIGLDIGSNSIGFAVVDANSHLLRLKGETAIGARLFREGQSAADRRGSRTTRRRLSRTRWRLSFLRDFFAPHITK IDPDFFLRQKYSEISPKDKDRFKYEKRLFNDRTDAEFYEDYPSMYHLRLHLMTHTHKADPREIFLAIHHILKSRGHFLTPGAA KDFNTDKVDLEDIFPALTEAYAQVYPDLELTFDLAKADDFKAKLLDEQATPSDTQKALVNLLLSSDGEKEIVKKRKQVLTEFA KAITGLKTKFNLALGTEVDEADASNWQFSMGQLDDKWSNIETSMTDQGTEIFEQIQELYRARLLNGIVPAGMSLSQAKVADYG QHKEDLELFKTYLKKLNDHELAKTIRGLYDRYINGDDAKPFLREDFVKALTKEVTAHPNEVSEQLLNRMGQANFMLKQRTKAN GAIPIQLQQRELDQIIANQSKYYDWLAAPNPVEAHRWKMPYQLDELLNFHIPYYVGPLITPKQQAESGENVFAWMVRKDPSGN ITPYNFDEKVDREASANTFIQRMKTTDTYLIGEDVLPKQSLLYQKYEVLNELNNVRINNECLGTDQKQRLIREVFERHSSVTI KQVADNLVAHGDFARRPEIRGLADEKRFLSSLSTYHQLKEILHEAIDDPTKLLDIENIITWSTVFEDHTIFETKLAEIEWLDP KKINELSGIRYRGWGQFSRKLLDGLKLGNGHTVIQELMLSNHNLMQILADETLKETMTELNQDKLKTDDIEDVINDAYTSPSN KKALRQVLRVVEDIKHAANGQDPSWLFIETADGTGTAGKRTQSRQKQIQTVYANAAQELIDSAVRGELEDKIADKASFTDRLV LYFMQGGRDIYTGAPLNIDQLSHYDIDHILPQSLIKDDSLDNRVLVNATINREKNNVFASTLFAGKMKATWRKWHEAGLISGR KLRNLMLRPDEIDKFAKGFVARQLVETRQIIKLTEQIAAAQYPNTKIIAVKAGLSHQLREELDFPKNRDVNHYHHAFDAFLAA RIGTYLLKRYPKLAPFFTYGEFAKVDVKKFREFNFIGALTHAKKNIIAKDTGEIVWDKERDIRELDRIYNFKRMLITHEVYFE TADLFKQTIYAAKDSKERGGSKQLIPKKQGYPTQVYGGYTQESGSYNALVRVAEADTTAYQVIKISAQNASKIASANLKSREK GKQLLNEIVVKQLAKRRKNWKPSANSFKIVIPRFGMGTLFQNAKYGLFMVNSDTYYRNYQELWLSRENQKLLKKLFSIKYEKT QMNHDALQVYKAIIDQVEKFFKLYDINQFRAKLSDAIERFEKLPINTDGNKIGKTETLRQILIGLQANGTRSNVKNLGIKTDL GLLQVGSGIKLDKDTQIVYQSPSGLFKRRIPLADL SEQ ID NO: 13 MTKEYYLGLDVGTNSVGWAVTDSQYNLCKFKKKDMWGIRLFESANTAKDRRLQRGNRRRLERKKQRIDLLQEIFSPEICKIDP TFFIRLNESRLHLEDKSNDFKYPLFIEKDYSDIEYYKEFPTIFHLRKHLIESEEKQDIRLIYLALHNIIKTRGHFLIDGDLQS AKQLRPILDTFLLSLQEEQNLSVSLSENQKDEYEEILKNRSIAKSEKVKKLKNLFEISDELEKEEKKAQSAVIENFCKFIVGN KGDVCKFLRVSKEELEIDSFSFSEGKYEDDIVKNLEEKVPEKVYLFEQMKAMYDWNILVDILETEEYISFAKVKQYEKHKTNL RLLRDIILKYCTKDEYNRMFNDEKEAGSYTAYVGKLKKNNKKYWIEKKRNPEEFYKSLGKLLDKIEPLKEDLEVLTMMIEECK NHTLLPIQKNKDNGVIPHQVHEVELKKILENAKKYYSFLTETDKDGYSVVQKIESIFRFRIPYYVGPLSTRHQEKGSNVWMVR KPGREDRIYPWNMEEIIDFEKSNENFITRMTNKCTYLIGEDVLPKHSLLYSKYMVLNELNNVKVRGKKLPTSLKQKVFEDLFE NKSKVTGKNLLEYLQIQDKDIQIDDLSGFDKDFKTSLKSYLDFKKQIFGEEIEKESIQNMIEDIIKWITIYGNDKEMLKRVIR ANYSNQLTEEQMKKITGFQYSGWGNFSKMFLKGISGSDVSTGETFDIITAMWETDNNLMQILSKKFTFMDNVEDFNSGKVGKI DKITYDSTVKEMFLSPENKRAVWQTIQVAEEIKKVMGCEPKKIFIEMARGGEKVKKRTKSRKAQLLELYAACEEDCRELIKEI EDRDERDFNSMKLFLYYTQFGKCMYSGDDIDINELIRGNSKWDRDHIYPQSKIKDDSIDNLVLVNKTYNAKKSNELLSEDIQK KMHSFWLSLLNKKLITKSKYDRLTRKGDFTDEELSGFIARQLVETRQSTKAIADIFKQIYSSEVVYVKSSLVSDFRKKPLNYL KSRRVNDYHHAKDAYLNIVVGNVYNKKFTSNPIQWMKKNRDTNYSLNKVFEHDVVINGEVIWEKCTYHEDTNTYDGGTLDRIR KIVERDNILYTEYAYCEKGELFNATIQNKNGNSTVSLKKGLDVKKYGGYFSANTSYFSLIEFEDKKGDRARHIIGVPIYIANM LEHSPSAFLEYCEQKGYQNVRILVEKIKKNSLLIINGYPLRIRGENEVDTSFKRAIQLKLDQKNYELVRNIEKFLEKYVEKKG NYPIDENRDHITHEKMNQLYEVLLSKMKKFNKKGMADPSDRIEKSKPKFIKLEDLIDKINVINKMLNLLRCDNDTKADLSLIE LPKNAGSFVVKKNTIGKSKIILVNQSVTGLYENRREL SEQ ID NO: 14 MARDYSVGLDIGTSSVGWAAIDNKYHLIRAKSKNLIGVRLFDSAVTAEKRRGYRTTRRRLSRRHWRLRLLNDIFAGPLTDFGD ENFLARLKYSWVHPQDQSNQAHFAAGLLFDSKEQDKDFYRKYPTIYHLRLALMNDDQKHDLREVYLAIHHLVKYRGHFLIEGD VKADSAFDVHTFADAIQRYAESNNSDENLLGKIDEKKLSAALTDKHGSKSQRAETAETAFDILDLQSKKQIQAILKSVVGNQA NLMAIFGLDSSAISKDEQKNYKFSFDDADIDEKIADSEALLSDTEFEFLCDLKAAFDGLTLKMLLGDDKTVSAAMVRRFNEHQ KDWEYIKSHIRNAKNAGNGLYEKSKKFDGINAAYLALQSDNEDDRKKAKKIFQDEISSADIPDDVKADFLKKIDDDQFLPIQR TKNNGTIPHQLHRNELEQIIEKQGIYYPFLKDTYQENSHELNKITALINFRVPYYVGPLVEEEQKIADDGKNIPDPTNHWMVR KSNDTITPWNLSQVVDLDKSGRRFIERLTGTDTYLIGEPTLPKNSLLYQKFDVLQELNNIRVSGRRLDIRAKQDAFEHLFKVQ KTVSATNLKDFLVQAGYISEDTQIEGLADVNGKNFNNALTTYNYLVSVLGREFVENPSNEELLEEITELQTVFEDKKVLRRQL DQLDGLSDHNREKLSRKHYTGWGRISKKLLTTKIVQNADKIDNQTFDVPRMNQSIIDTLYNTKMNLMEIINNAEDDFGVRAWI DKQNTTDGDEQDVYSLIDELAGPKEIKRGIVQSFRILDDITKAVGYAPKRVYLEFARKTQESHLTNSRKNQLSTLLKNAGLSE LVTQVSQYDAAALQNDRLYLYFLQQGKDMYSGEKLNLDNLSNYDIDHIIPQAYTKDNSLDNRVLVSNITNRRKSDSSNYLPAL IDKMRPFWSVLSKQGLLSKHKFANLTRTRDFDDMEKERFIARSLVETRQIIKNVASLIDSHFGGETKAVAIRSSLTADMRRYV DIPKNRDINDYHHAFDALLFSTVGQYTENSGLMKKGQLSDSAGNQYNRYIKEWIHAARLNAQSQRVNPFGFVVGSMRNAAPGK LNPETGEITPEENADWSIADLDYLHKVMNFRKITVTRRLKDQKGQLYDESRYPSVLHDAKSKASINFDKHKPVDLYGGFSSAK PAYAALIKFKNKFRLVNVLRQWTYSDKNSEDYILEQIRGKYPKAEMVLSHIPYGQLVKKDGALVTISSATELHNFEQLWLPLA DYKLINTLLKTKEDNLVDILHNRLDLPEMTIESAFYKAFDSILSFAFNRYALHQNALVKLQAHRDDFNALNYEDKQQTLERIL DALHASPASSDLKKINLSSGFGRLFSPSHFTLADTDEFIFQSVTGLFSTQKTVAQLYQETK SEQ ID NO: 15 MVYDVGLDIGTGSVGWVALDENGKLARAKGKNLVGVRLFDTAQTAADRRGFRTTRRRLSRRKWRLRLLDELFSAEINEIDSSF FQRLKYSYVHPKDEENKAHYYGGYLFPTEEETKKFHRSYPTIYHLRQELMAQPNKRFDIREIYLAIHHLVKYRGHFLSSQEKI TIGSTYNPEDLANAIEVYADEKGLSWELNNPEQLTEIISGEAGYGLNKSMKADEALKLFEFDNNQDKVAIKTLLAGLTGNQID FAKLFGKDISDKDEAKLWKLKLDDEALEEKSQTILSQLTDEEIELFHAVVQAYDGFVLIGLLNGADSVSAAMVQLYDQHREDR KLLKSLAQKAGLKHKRFSEIYEQLALATDEATIKNGISTARELVEESNLSKEVKEDTLRRLDENEFLPKQRTKANSVIPHQLH LAELQKILQNQGQYYPFLLDTFEKEDGQDNKIEELLRFRIPYYVGPLVTKKDVEHAGGDADNHWVERNEGFEKSRVTPWNFDK VFNRDKAARDFIERLTGNDTYLIGEKTLPQNSLRYQLFTVLNELNNVRVNGKKFDSKTKADLINDLFKARKTVSLSALKDYLK AQGKGDVTITGLADESKFNSSLSSYNDLKKTFDAEYLENEDNQETLEKIIEIQTVFEDSKIASRELSKLPLDDDQVKKLSQTH YTGWGRLSEKLLDSKIIDERGQKVSILDKLKSTSQNFMSIINNDKYGVQAWITEQNTGSSKLTFDEKVNELTTSPANKRGIKQ SFAVLNDIKKAMKEEPRRVYLEFAREDQTSVRSVPRYNQLKEKYQSKSLSEEAKVLKKTLDGNKNKMSDDRYFLYFQQQGKDM YTGRPINFERLSQDYDIDHIIPQAFTKDDSLDNRVLVSRPENARKSDSFAYTDEVQKQDGSLWTSLLKSGFINRKKYERLTKA GKYLDGQKTGFIARQLVETRQIIKNVASLIEGEYENSKAVAIRSEITADMRLLVGIKKHREINSFHHAFDALLITAAGQYMQN RYPDRDSTNVYNEFDRYTNDYLKNLRQLSSRDEVRRLKSFGFVVGTMRKGNEDWSEENTSYLRKVMMFKNILTTKKTEKDRGP LNKETIFSPKSGKKLIPLNSKRSDTALYGGYSNVYSAYMTLVRANGKNLLIKIPISIANQIEVGNLKINDYIVNNPAIKKFEK ILISKLPLGQLVNEDGNLIYLASNEYRHNAKQLWLSTTDADKIASISENSSDEELLEAYDILTSENVKNRFPFFKKDIDKLSQ VRDEFLDSDKRIAVIQTILRGLQIDAAYQAPVKIISKKVSDWHKLQQSGGIKLSDNSEMIYQSATGIFETRVKISDLL SEQ ID NO: 16 IVDYCIGLDLGTGSVGWAVVDMNHRLMKRNGKHLWGSRLFSNAETAANRRASRSIRRRYNKRRERIRLLRAILQDMVLEKDPT FFIRLEHTSFLDEEDKAKYLGTDYKDNYNLFIDEDFNDYTYYHKYPTIYHLRKALCESTEKADPRLIYLALHHIVKYRGNFLY EGQKFNMDASNIEDKLSDIFTQFTSFNNIPYEDDEKKNLEILEILKKPLSKKAKVDEVMTLIAPEKDYKSAFKELVTGIAGNK MNVTKMILCEPIKQGDSEIKLKFSDSNYDDQFSEVEKDLGEYVEFVDALHNVYSWVELQTIMGATHTDNASISEAMVSRYNKH HDDLKLLKDCIKNNVPNKYFDMFRNDSEKSKGYYNYINRPSKAPVDEFYKYVKKCIEKVDTPEAKQILNDIELENFLLKQNSR TNGSVPYQMQLDEMIKIIDNQAEYYPILKEKREQLLSILTFRIPYYFGPLNETSEHAWIKRLEGKENQRILPWNYQDIVDVDA TAEGFIKRMRSYCTYFPDEEVLPKNSLIVSKYEVYNELNKIRVDDKLLEVDVKNDIYNELFMKNKTVTEKKLKNWLVNNQCCS KDAEIKGFQKENQFSTSLTPWIDFTNIFGKIDQSNFDLIENIIYDLTVFEDKKIMKRRLKKKYALPDDKVKQILKLKYKDWSR LSKKLLDGIVADNRFGSSVTVLDVLEMSRLNLMEIINDKDLGYAQMIEEATSCPEDGKFTYEEVERLAGSPALKRGIWQSLQI VEEITKVMKCRPKYIYIEFERSEEAKERTESKIKKLENVYKDLDEQTKKEYKSVLEELKGFDNTKKISSDSLFLYFTQLGKCM YSGKKLDIDSLDKYQIDHIVPQSLVKDDSFDNRVLVVPSENQRKLDDLVVPFDIRDKMYRFWKLLFDHELISPKKFYSLIKTE YTERDEERFINRQLVETRQITKNVTQIIEDHYSTTKVAAIRANLSHEFRVKNHIYKNRDINDYHHAHDAYIVALIGGFMRDRY PNMHDSKAVYSEYMKMFRKNKNDQKRWKDGFVINSMNYPYEVDGKLIWNPDLINEIKKCFYYKDCYCTTKLDQKSGQLFNLTV LSNDAHADKGVTKAVVPVNKNRSDVHKYGGFSGLQYTIVAIEGQKKKGKKTELVKKISGVPLHLKAASINEKINYIEEKEGLS DVRIIKDNIPVNQMIEMDGGEYLLTSPTEYVNARQLVLNEKQCALIADIYNAIYKQDYDNLDDILMIQLYIELTNKMKVLYPA YRGIAEKFESMNENYVVISKEEKANIIKQMLIVMHRGPQNGNIVYDDFKISDRIGRLKTKNHNLNNIVFISQSPTGIYTKKYK L SEQ ID NO: 17 MKSEKKYYIGLDVGTNSVGWAVTDEFYNILRAKGKDLWGVRLFEKADTAANTRIFRSGRRRNDRKGMRLQILREIFEDEIKKV DKDFYDRLDESKFWAEDKKVSGKYSLFNDKNFSDKQYFEKFPTIFHLRKYLMEEHGKVDIRYYFLAINQMMKRRGHFLIDGQI SHVTDDKPLKEQLILLINDLLKIELEEELMDSIFEILADVNEKRTDKKNNLKELIKGQDFNKQEGNILNSIFESIVTGKAKIK NIISDEDILEKIKEDNKEDFVLTGDSYEENLQYFEEVLQENITLFNTLKSTYDFLILQSILKGKSTLSDAQVERYDEHKKDLE ILKKVIKKYDEDGKLFKQVFKEDNGNGYVSYIGYYLNKNKKITAKKKISNIEFTKYVKGILEKQCDCEDEDVKYLLGKIEQEN FLLKQISSINSVIPHQIHLFELDKILENLAKNYPSFNNKKEEFTKIEKIRKTFTFRIPYYVGPLNDYHKNNGGNAWIFRNKGE KIRPWNFEKIVDLHKSEEEFIKRMLNQCTYLPEETVLPKSSILYSEYMVLNELNNLRINGKPLDTDVKLKLIEELFKKKTKVT LKSIRDYMVRNNFADKEDFDNSEKNLEIASNMKSYIDFNNILEDKFDVEMVEDLIEKITIHTGNKKLLKKYIEETYPDLSSSQ IQKIINLKYKDWGRLSRKLLDGIKGTKKETEKTDTVINFLRNSSDNLMQIIGSQNYSFNEYIDKLRKKYIPQEISYEVVENLY VSPSVKKMIWQVIRVTEEITKVMGYDPDKIFIEMAKSEEEKKTTISRKNKLLDLYKAIKKDERDSQYEKLLTGLNKLDDSDLR SRKLYLYYTQMGRDMYTGEKIDLDKLFDSTHYDKDHIIPQSMKKDDSIINNLVLVNKNANQTTKGNIYPVPSSIRNNPKIYNY WKYLMEKEFISKEKYNRLIRNTPLTNEELGGFINRQLVETRQSTKAIKELFEKFYQKSKIIPVKASLASDLRKDMNTLKSREV NDLHHAHDAFLNIVAGDVWNREFTSNPINYVKENREGDKVKYSLSKDFTRPRKSKGKVIWTPEKGRKLIVDTLNKPSVLISNE SHVKKGELFNATIAGKKDYKKGKIYLPLKKDDRLQDVSKYGGYKAINGAFFFLVEHTKSKKRIRSIELFPLHLLSKFYEDKNT VLDYAINVLQLQDPKIIIDKINYRTEIIIDNFSYLISTKSNDGSITVKPNEQMYWRVDEISNLKKIENKYKKDAILTEEDRKI MESYIDKIYQQFKAGKYKNRRTTDTIIEKYEIIDLDTLDNKQLYQLLVAFISLSYKTSNNAVDFTVIGLGTECGKPRITNLPD NTYLVYKSITGIYEKRIRIK SEQ ID NO: 18 MKLRGIEDDYSIGLDMGTSSVGWAVTDERGTLAHFKRKPTWGSRLFREAQTAAVARMPRGQRRRYVRRRWRLDLLQKLFEQQM EQADPDFFIRLRQSRLLRDDRAEEHADYRWPLFNDCKFTERDYYQRFPTIYHVRSWLMETDEQADIRLIYLALHNIVKHRGNF LREGQSLSAKSARPDEALNHLRETLRVWSSERGFECSIADNGSILAMLTHPDLSPSDRRKKIAPLFDVKSDDAAADKKLGIAL AGAVIGLKTEFKNIFGDFPCEDSSIYLSNDEAVDAVRSACPDDCAELFDRLCEVYSAYVLQGLLSYAPGQTISANMVEKYRRY GEDLALLKKLVKIYAPDQYRMFFSGATYPGTGIYDAAQARGYTKYNLGPKKSEYKPSESMQYDDFRKAVEKLFAKTDARADER YRMMMDRFDKQQFLRRLKTSDNGSIYHQLHLEELKAIVENQGRFYPFLKRDADKLVSLVSFRIPYYVGPLSTRNARTDQHGEN RFAWSERKPGMQDEPIFPWNWESIIDRSKSAEKFILRMTGMCTYLQQEPVLPKSSLLYEEFCVLNELNGAHWSIDGDDEHRFD AADREGIIEELFRRKRTVSYGDVAGWMERERNQIGAHVCGGQGEKGFESKLGSYIFFCKDVFKVERLEQSDYPMIERIILWNT LFEDRKILSQRLKEEYGSRLSAEQIKTICKKRFTGWGRLSEKFLTGITVQVDEDSVSIMDVLREGCPVSGKRGRAMVMMEILR DEELGFQKKVDDFNRAFFAENAQALGVNELPGSPAVRRSLNQSIRIVDEIASIAGKAPANIFIEVTRDEDPKKKGRRTKRRYN DLKDALEAFKKEDPELWRELCETAPNDMDERLSLYFMQRGKCLYSGRAIDIHQLSNAGIYEVDHIIPRTYVKDDSLENKALVY REENQRKTDMLLIDPEIRRRMSGYWRMLHEAKLIGDKKFRNLLRSRIDDKALKGFIARQLVETGQMVKLVRSLLEARYPETNI ISVKASISHDLRTAAELVKCREANDFHHAHDAFLACRVGLFIQKRHPCVYENPIGLSQVVRNYVRQQADIFKRCRTIPGSSGF IVNSFMTSGFDKETGEIFKDDWDAEAEVEGIRRSLNFRQCFISRMPFEDHGVFWDATIYSPRAKKTAALPLKQGLNPSRYGSF SREQFAYFFIYKARNPRKEQTLFEFAQVPVRLSAQIRQDENALERYARELAKDQGLEFIRIERSKILKNQLIEIDGDRLCITG KEEVRNACELAFAQDEMRVIRMLVSEKPVSRECVISLFNRILLHGDQASRRLSKQLKLALLSEAFSEASDNVQRNVVLGLIAI FNGSTNMVNLSDIGGSKFAGNVRIKYKKELASPKVNVHLIDQSVTGMFERRTKIGL SEQ ID NO: 19 MENKQYYIGLDVGTNSVGWAVTDTSYNLLRAKGKDMWGARLFEKANTAAERRTKRTSRRRSEREKARKAMLKELFADEINRVD PSFFIRLEESKFFLDDRSENNRQRYTLFNDATFTDKDYYEKYKTIFHLRSALINSDEKFDVRLVFLAILNLFSHRGHFLNASL KGDGDIQGMDVFYNDLVESCEYFEIELPRITNIDNFEKILSQKGKSRTKILEELSEELSISKKDKSKYNLIKLISGLEASVVE LYNIEDIQDENKKIKIGFRESDYEESSLKVKEIIGDEYFDLVERAKSVHDMGLLSNIIGNSKYLCEARVEAYENHHKDLLKIK ELLKKYDKKAYNDMFRKMTDKNYSAYVGSVNSNIAKERRSVDKRKIEDLYKYIEDTALKNIPDDNKDKIEILEKIKLGEFLKK QLTASNGVIPNQLQSRELRAILKKAENYLPFLKEKGEKNLTVSEMIIQLFEFQIPYYVGPLDKNPKKDNKANSWAKIKQGGRI LPWNFEDKVDVKGSRKEFIEKMVRKCTYISDEHTLPKQSLLYEKFMVLNEINNIKIDGEKISVEAKQKIYNDLFVKGKKVSQK DIKKELISLNIMDKDSVLSGTDTVCNAYLSSIGKFTGVFKEEINKQSIVDMIEDIIFLKTVYGDEKRFVKEEIVEKYGDEIDK DKIKRILGFKFSNWGNLSKSFLELEGADVGTGEVRSIIQSLWETNFNLMELLSSRFTYMDELEKRVKKLEKPLSEWTIEDLDD MYLSSPVKRMIWQSMKIVDEIQTVIGYAPKRIFVEMTRSEGEKVRTKSRKDRLKELYNGIKEDSKQWVKELDSKDESYFRSKK MYLYYLQKGRCMYSGEVIELDKLMDDNLYDIDHIYPRSFVKDDSLDNLVLVKKEINNRKQNDPITPQIQASCQGFWKILHDQG FMSNEKYSRLTRKTQEFSDEEKLSFINRQIVETGQATKCMAQILQKSMGEDVDVVFSKARLVSEFRHKFELFKSRLINDFHHA NDAYLNIVVGNSYFVKFTRNPANFIKDARKNPDNPVYKYHMDRFFERDVKSKSEVAWIGQSEGNSGTIVIVKKTMAKNSPLIT KKVEEGHGSITKETIVGVKEIKFGRNKVEKADKTPKKPNLQAYRPIKTSDERLCNILRYGGRTSISISGYCLVEYVKKRKTIR SLEAIPVYLGRKDSLSEEKLLNYFRYNLNDGGKDSVSDIRLCLPFISTNSLVKIDGYLYYLGGKNDDRIQLYNAYQLKMKKEE VEYIRKIEKAVSMSKFDEIDREKNPVLTEEKNIELYNKIQDKFENTVFSKRMSLVKYNKKDLSFGDFLKNKKSKFEEIDLEKQ CKVLYNIIFNLSNLKEVDLSDIGGSKSTGKCRCKKNITNYKEFKLIQQSITGLYSCEKDLMTI SEQ ID NO: 20 MKNLKEYYIGLDIGTASVGWAVTDESYNIPKFNGKKMWGVRLFDDAKTAEERRTQRGSRRRLNRRKERINLLQDLFATEISKV DPNFFLRLDNSDLYREDKDEKLKSKYTLFNDKDFKDRDYHKKYPTIHHLIMDLIEDEGKKDIRLLYLACHYLLKNRGHFIFEG QKFDTKNSFDKSINDLKIHLRDEYNIDLEFNNEDLIEIITDTTLNKTNKKKELKNIVGDTKFLKAISAIMIGSSQKLVDLFED GEFEETTVKSVDFSTTAFDDKYSEYEEALGDTISLLNILKSIYDSSILENLLKDADKSKDGNKYISKAFVKKFNKHGKDLKTL KRIIKKYLPSEYANIFRNKSINDNYVAYTKSNITSNKRTKASKFTKQEDFYKFIKKHLDTIKETKLNSSENEDLKLIDEMLTD IEFKTFIPKLKSSDNGVIPYQLKLMELKKILDNQSKYYDFLNESDEYGTVKDKVESIMEFRIPYYVGPLNPDSKYAWIKRENT KITPWNFKDIVDLDSSREEFIDRLIGRCTYLKEEKVLPKASLIYNEFMVLNELNNLKLNEFLITEEMKKAIFEELFKTKKKVT LKAVSNLLKKEFNLTGDILLSGTDGDFKQGLNSYIDFKNIIGDKVDRDDYRIKIEEIIKLIVLYEDDKTYLKKKIKSAYKNDF TDDEIKKIAALNYKDWGRLSKRFLTGIEGVDKTTGEKGSIIYFMREYNLNLMELMSGHYTFTEEVEKLNPVENRELCYEMVDE LYLSPSVKRMLWQSLRVVDEIKRIIGKDPKKIFIEMARAKEAKNSRKESRKNKLLEFYKFGKKAFINEIGEERYNYLLNEINS EEESKFRWDNLYLYYTQLGRCMYSLEPIDLADLKSNNIYDQDHIYPKSKIYDDSLENRVLVKKNLNHEKGNQYPIPEKVLNKN AYGFWKILFDKGLIGQKKYTRLTRRTPFEERELAEFIERQIVETRQATKETANLLKNICQDSEIVYSKAENASRFRQEFDIIK CRTVNDLHHMHDAYLNIVVGNVYNTKFTKNPLNFIKDKDNVRSYNLENMFKYDVVRGSYTAWIADDSEGNVKAATIKKVKREL EGKNYRFTRMSYIGTGGLYDQNLMRKGKGQIPQKENTNKSNIEKYGGYNKASSAYFALIESDGKAGRERTLETIPIMVYNQEK YGNTEAVDKYLKDNLELQDPKILKDKIKINSLIKLDGFLYNIKGKTGDSLSIAGSVQLIVNKEEQKLIKKMDKFLVKKKDNKD IKVTSFDNIKEEELIKLYKTLSDKLNNGIYSNKRNNQAKNISEALDKFKEISIEEKIDVLNQIILLFQSYNNGCNLKSIGLSA KTGVVFIPKKLNYKECKLINQSITGLFENEVDLLNL SEQ ID NO: 21 MGKMYYLGLDIGTNSVGYAVTDPSYHLLKFKGEPMWGAHVFAAGNQSAERRSFRTSRRRLDRRQQRVKLVQEIFAPVISPIDP RFFIRLHESALWRDDVAETDKHIFFNDPTYTDKEYYSDYPTIHHLIVDLMESSEKHDPRLVYLAVAWLVAHRGHFLNEVDKDN IGDVLSFDAFYPEFLAFLSDNGVSPWVCESKALQATLLSRNSVNDKYKALKSLIFGSQKPEDNFDANISEDGLIQLLAGKKVK VNKLFPQESNDASFTLNDKEDAIEEILGTLTPDECEWIAHIRRLFDWAIMKHALKDGRTISESKVKLYEQHHHDLTQLKYFVK TYLAKEYDDIFRNVDSETTKNYVAYSYHVKEVKGTLPKNKATQEEFCKYVLGKVKNIECSEADKVDFDEMIQRLTDNSFMPKQ VSGENRVIPYQLYYYELKTILNKAASYLPFLTQCGKDAISNQDKLLSIMTFRIPYFVGPLRKDNSEHAWLERKAGKIYPWNFN DKVDLDKSEEAFIRRMTNTCTYYPGEDVLPLDSLIYEKFMILNEINNIRIDGYPISVDVKQQVFGLFEKKRRVTVKDIQNLLL SLGALDKHGKLTGIDTTIHSNYNTYHHFKSLMERGVLTRDDVERIVERMTYSDDTKRVRLWLNNNYGTLTADDVKHISRLRKH DFGRLSKMFLTGLKGVHKETGERASILDFMWNTNDNLMQLLSECYTFSDEITKLQEAYYAKAQLSLNDFLDSMYISNAVKRPI YRTLAVVNDIRKACGTAPKRIFIEMARDGESKKKRSVTRREQIKNLYRSIRKDFQQEVDFLEKILENKSDGQLQSDALYLYFA QLGRDMYTGDPIKLEHIKDQSFYNIDHIYPQSMVKDDSLDNKVLVQSEINGEKSSRYPLDAAIRNKMKPLWDAYYNHGLISLK KYQRLTRSTPFTDDEKWDFINRQLVETRQSTKALAILLKRKFPDTEIVYSKAGLSSDFRHEFGLVKSRNINDLHHAKDAFLAI VTGNVYHERFNRRWFMVNQPYSVKTKTLFTHSIKNGNFVAWNGEEDLGRIVKMLKQNKNTIHFTRFSFDRKEGLFDIQPLKAS TGLVPRKAGLDVVKYGGYDKSTAAYYLLVRFTLEDKKTQHKLMMIPVEGLYKARIDHDKEFLTDYAQTTISEILQKDKQKVIN IMFPMGTRHIKLNSMISIDGFYLSIGGKSSKGKSVLCHAMVPLIVPHKIECYIKAMESFARKFKENNKLRIVEKFDKITVEDN LNLYELFLQKLQHNPYNKFFSTQFDVLTNGRSTFTKLSPEEQVQTLLNILSIFKTCRSSGCDLKSINGSAQAARIMISADLTG LSKKYSDIRLVEQSASGLFVSKSQNLLEYL SEQ ID NO: 22 MTKKEQPYNIGLDIGTSSVGWAVTNDNYDLLNIKKKNLWGVRLFEEAQTAKETRLNRSTRRRYRRRKNRINWLNEIFSEELAK TDPSFLIRLQNSWVSKKDPDRKRDKYNLFIDGPYTDKEYYREFPTIFHLRKELILNKDKADIRLIYLALHNILKYRGNFTYEH QKFNISNLNNNLSKELIELNQQLIKYDISFPDDCDWNHISDILIGRGNATQKSSNILKDFTLDKETKKLLKEVINLILGNVAH LNTIFKTSLTKDEEKLNFSGKDIESKLDDLDSILDDDQFTVLDAANRIYSTITLNEILNGESYFSMAKVNQYENHAIDLCKLR DMWHTTKNEEAVEQSRQAYDDYINKPKYGTKELYTSLKKFLKVALPTNLAKEAEEKISKGTYLVKPRNSENGVVPYQLNKIEM EKIIDNQSQYYPFLKENKEKLLSILSFRIPYYVGPLQSAEKNPFAWMERKSNGHARPWNFDEIVDREKSSNKFIRRMTVTDSY LVGEPVLPKNSLIYQRYEVLNELNNIRITENLKTNPIGSRLTVETKQRIYNELFKKYKKVTVKKLTKWLIAQGYYKNPILIGL SQKDEFNSTLTTYLDMKKIFGSSFMEDNKNYDQIEELIEWLTIFEDKQILNEKLHSSKYSYTPDQIKKISNMRYKGWGRLSKK ILMDITTETNTPQLLQLSNYSILDLMWATNNNFISIMSNDKYDFKNYIENHNLNKNEDQNISDLVNDIHVSPALKRGITQSIK IVQEIVKFMGHAPKHIFIEVTRETKKSEITTSREKRIKRLQSKLLNKANDFKPQLREYLVPNKKIQEELKKHKNDLSSERIML YFLQNGKSLYSEESLNINKLSDYQVDHILPRTYIPDDSLENKALVLAKENQRKADDLLLNSNVIDRNLERWTYMLNNNMIGLK KFKNLTRRVITDKDKLGFIHRQLVQTSQMVKGVANILDNMYKNQGTTCIQARANLSTAFRKALSGQDDTYHFKHPELVKNRNV NDFHHAQDAYLASFLGTYRLRRFPTNEMLLMNGEYNKFYGQVKELYSKKKKLPDSRKNGFIISPLVNGTTQYDRNTGEIIWNV GFRDKILKIFNYHQCNVTRKTEIKTGQFYDQTIYSPKNPKYKKLIAQKKDMDPNIYGGFSGDNKSSITIVKIDNNKIKPVAIP IRLINDLKDKKTLQNWLEENVKHKKSIQIIKNNVPIGQIIYSKKVGLLSLNSDREVANRQQLILPPEHSALLRLLQIPDEDLD QILAFYDKNILVEILQELITKMKKFYPFYKGEREFLIANIENFNQATTSEKVNSLEELITLLHANSTSAHLIFNNIEKKAFGR KTHGLTLNNTDFIYQSVTGLYETRIHIE SEQ ID NO: 23 MTKFNKNYSIGLDIGVSSVGYAVVTEDYRVPAFKFKVLGNTEKEKIKKNLIGSTTFVSAQPAKGTRVFRVNRRRIDRRNHRIT YLRDIFQKEIEKVDKNFYRRLDESFRVLGDKSEDLQIKQPFFGDKELETAYHKKYPTIYHLRKHLADADKNSPVADIREVYMA ISHILKYRGHFLTLDKINPNNINMQNSWIDFIESCQEVFDLEISDESKNIADIFKSSENRQEKVKKILPYFQQELLKKDKSIF KQLLQLLFGLKTKFKDCFELEEEPDLNFSKENYDENLENFLGSLEEDFSDVFAKLKVLRDTILLSGMLTYTGATHARFSATMV ERYEEHRKDLQRFKFFIKQNLSEQDYLDIFGRKTQNGFDVDKETKGYVGYITNKMVLTNPQKQKTIQQNFYDYISGKITGIEG AEYFLNKISDGTFLRKLRTSDNGAIPNQIHAYELEKIIERQGKDYPFLLENKDKLLSILTFKIPYYVGPLAKGSNSRFAWIKR ATSSDILDDNDEDTRNGKIRPWNYQKLINMDETRDAFITNLIGNDIILLNEKVLPKRSLIYEEVMLQNELTRVKYKDKYGKAH FFDSELRQNIINGLFKNNSKRVNAKSLIKYLSDNHKDLNAIEIVSGVEKGKSFNSTLKTYNDLKTIFSEELLDSEIYQKELEE IIKVITVFDDKKSIKNYLTKFFGHLEILDEEKINQLSKLRYSGWGRYSAKLLLDIRDEDTGFNLLQFLRNDEENRNLTKLISD NTLSFEPKIKDIQSKSTIEDDIFDEIKKLAGSPAIKRGILNSIKIVDELVQIIGYPPHNIVIEMARENMTTEEGQKKAKTRKT KLESALKNIENSLLENGKVPHSDEQLQSEKLYLYYLQNGKDMYTLDKTGSPAPLYLDQLDQYEVDHIIPYSFLPIDSIDNKVL THRENNQQKLNNIPDKETVANMKPFWEKLYNAKLISQTKYQRLTTSERTPDGVLTESMKAGFIERQLVETRQIIKHVARILDN RFSDTKIITLKSQLITNFRNTFHIAKIRELNDYHHAHDAYLAVVVGQTLLKVYPKLAPELIYGHHAHFNRHEENKATLRKHLY SNIMRFFNNPDSKVSKDIWDCNRDLPIIKDVIYNSQINFVKRTMIKKGAFYNQNPVGKFNKQLAANNRYPLKTKALCLDTSIY GGYGPMNSALSIIIIAERFNEKKGKIETVKEFHDIFIIDYEKFNNNPFQFLNDTSENGFLKKNNINRVLGFYRIPKYSLMQKI DGTRMLFESKSNLHKATQFKLTKTQNELFFHMKRLLTKSNLMDLKSKSAIKESQNFILKHKEEFDNISNQLSAFSQKMLGNTT SLKNLIKGYNERKIKEIDIRDETIKYFYDNFIKMFSFVKSGAPKDINDFFDNKCTVARMRPKPDKKLLNATLIHQSITGLYET RIDLSKLGED SEQ ID NO: 24 MKQEYFLGLDMGTGSLGWAVTDSTYQVMRKHGKALWGTRLFESASTAEERRMFRTARRRLDRRNWRIQVLQEIFSEEISKVDP GFFLRMKESKYYPEDKRDAEGNCPELPYALFVDDNYTDKNYHKDYPTIYHLRKMLMETTEIPDIRLVYLVLHHMMKHRGHFLL SGDISQIKEFKSTFEQLIQNIQDEELEWHISLDDAAIQFVEHVLKDRNLTRSTKKSRLIKQLNAKSACEKAILNLLSGGTVKL SDIFNNKELDESERPKVSFADSGYDDYIGIVEAELAEQYYIIASAKAVYDWSVLVEILGNSVSISEAKIKVYQKHQADLKTLK KIVRQYMTKEDYKRVFVDTEEKLNNYSAYIGMTKKNGKKVDLKSKQCTQADFYDFLKKNVIKVIDHKEITQEIESEIEKENFL PKQVTKDNGVIPYQVHDYELKKILDNLGTRMPFIKENAEKIQQLFEFRIPYYVGPLNRVDDGKDGKFTWSVRKSDARIYPWNF TEVIDVEASAEKFIRRMTNKCTYLVGEDVLPKDSLVYSKFMVLNELNNLRLNGEKISVELKQRIYEELFCKYRKVTRKKLERY LVIEGIAKKGVEITGIDGDFKASLTAYHDFKERLTDVQLSQRAKEAIVLNVVLFGDDKKLLKQRLSKMYPNLTTGQLKGICSL SYQGWGRLSKTFLEEITVPAPGTGEVWNIMTALWQTNDNLMQLLSRNYGFTNEVEEFNTLKKETDLSYKTVDELYVSPAVKRQ IWQTLKVVKEIQKVMGNAPKRVFVEMAREKQEGKRSDSRKKQLVELYRACKNEERDWITELNAQSDQQLRSDKLFLYYIQKGR CMYSGETIQLDELWDNTKYDIDHIYPQSKTMDDSLNNRVLVKKNYNAIKSDTYPLSLDIQKKMMSFWKMLQQQGFITKEKYVR LVRSDELSADELAGFIERQIVETRQSTKAVATILKEALPDTEIVYVKAGNVSNFRQTYELLKVREMNDLHHAKDAYLNIVVGN AYFVKFTKNAAWFIRNNPGRSYNLKRMFEFDIERSGEIAWKAGNKGSIVTVKKVMQKNNILVTRKAYEVKGGLFDQQIMKKGK GQVPIKGNDERLADIEKYGGYNKAAGTYFMLVKSLDKKGKEIRTIEFVPLYLKNQIEINHESAIQYLAQERGLNSPEILLSKI KIDTLFKVDGFKMWLSGRTGNQLIFKGANQLILSHQEAAILKGVVKYVNRKNENKDAKLSERDGMTEEKLLQLYDTFLDKLSN TVYSIRLSAQIKTLTEKRAKFIGLSNEDQCIVLNEILHMFQCQSGSANLKLIGGPGSAGILVMNNNITACKQISVINQSPTGI YEKEIDLIKL SEQ ID NO: 25 MKKPYSIGLDIGTNSVGWAVVTDDYKVPAKKMKVLGNTDKSHIEKNLLGALLFDSGNTAEDRRLKRTARRRYTRRRNRILYLQ EIFSEEMGKVDDSFFHRLEDSFLVTEDKRGERHPIFGNLEEEVKYHENFPTIYHLRQYLADNPEKVDLRLVYLALAHIIKFRG HFLIEGKFDTRNNDVQRLFQEFLAVYDNTFENSSLQEQNVQVEEILTDKISKSAKKDRVLKLFPNEKSNGRFAEFLKLIVGNQ ADFKKHFELEEKAPLQFSKDTYEEELEVLLAQIGDNYAELFLSAKKLYDSILLSGILTVTDVGTKAPLSASMIQRYNEHQMDL AQLKQFIRQKLSDKYNEVFSDVSKDGYAGYIDGKTNQEAFYKYLKGLLNKIEGSGYFLDKIEREDFLRKQRTFDNGSIPHQIH LQEMRAIIRRQAEFYPFLADNQDRIEKLLTFRIPYYVGPLARGKSDFAWLSRKSADKITPWNFDEIVDKESSAEAFINRMTNY DLYLPNQKVLPKHSLLYEKFTVYNELTKVKYKTEQGKTAFFDANMKQEIFDGVFKVYRKVTKDKLMDFLEKEFDEFRIVDLTG LDKENKVFNASYGTYHDLCKILDKDFLDNSKNEKILEDIVLTLTLFEDREMIRKRLENYSDLLTKEQVKKLERRHYTGWGRLS AELIHGIRNKESRKTILDYLIDDGNSNRNFMQLINDDALSFKEEIAKAQVIGETDNLNQVVSDIAGSPAIKKGILQSLKIVDE LVKIMGHQPENIVVEMARENQFTNQGRRNSQQRLKGLTDSIKEFGSQILKEHPVENSQLQNDRLFLYYLQNGRDMYTGEELDI DYLSQYDIDHIIPQAFIKDNSIDNRVLTSSKENRGKSDDVPSKDVVRKMKSYWSKLLSAKLITQRKFDNLTKAERGGLTDDDK AGFIKRQLVETRQITKHVARILDERFNTETDENNKKIRQVKIVTLKSNLVSNFRKEFELYKVREINDYHHAHDAYLNAVIGKA LLGVYPQLEPEFVYGDYPHFHGHKENKATAKKFFYSNIMNFFKKDDVRTDKNGEIIWKKDEHISNIKKVLSYPQVNIVKKVEE QTGGFSKESILPKGNSDKLIPRKTKKFYWDTKKYGGFDSPIVAYSILVIADIEKGKSKKLKTVKALVGVTIMEKMTFERDPVA FLERKGYRNVQEENIIKLPKYSLFKLENGRKRLLASARELQKGNEIVLPNHLGTLLYHAKNIHKVDEPKHLDYVDKHKDEFKE LLDVVSNFSKKYTLAEGNLEKIKELYAQNNGEDLKELASSFINLLTFTAIGAPATFKFFDKNIDRKRYTSTTEILNATLIHQS ITGLYETRIDLNKLGGD SEQ ID NO: 26 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQ EIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRG HFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLT PNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDL TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIH LGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNF DKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEIS GVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSR KLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDI NRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTA LIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRD FATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKE LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKL KGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFD TTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SEQ ID NO: 27 MTKPYSIGLDIGTNSVGWAVTTDNYKVPSKKMKVLGNTSKKYIKKNLLGVLLFDSGITAEGRRLKRTARRRYTRRRNRILYLQ EIFSTEMATLDDAFFQRLDDSFLVPDDKRDSKYPIFGNLVEEKAYHDEFPTIYHLRKYLADSTKKADLRLVYLALAHMIKYRG HFLIEGEFNSKNNDIQKNFQDFLDTYNAIFESDLSLENSKQLEEIVKDKISKLEKKDRILKLFPGEKNSGIFSEFLKLIVGNQ ADFRKCFNLDEKASLHFSKESYDEDLETLLGYIGDDYSDVFLKAKKLYDAILLSGFLTVTDNETEAPLSSAMIKRYNEHKEDL ALLKEYIRNISLKTYNEVFKDDTKNGYAGYIDGKTNQEDFYVYLKKLLAEFEGADYFLEKIDREDFLRKQRTFDNGSIPYQIH LQEMRAILDKQAKFYPFLAKNKERIEKILTFRIPYYVGPLARGNSDFAWSIRKRNEKITPWNFEDVIDKESSAEAFINRMTSF DLYLPEEKVLPKHSLLYETFNVYNELTKVRFIAESMRDYQFLDSKQKKDIVRLYFKDKRKVTDKDIIEYLHAIYGYDGIELKG IEKQFNSSLSTYHDLLNIINDKEFLDDSSNEAIIEEIIHTLTIFEDREMIKQRLSKFENIFDKSVLKKLSRRHYTGWGKLSAK LINGIRDEKSGNTILDYLIDDGISNRNFMQLIHDDALSFKKKIQKAQIIGDEDKGNIKEVVKSLPGSPAIKKGILQSIKIVDE LVKVMGGRKPESIVVEMARENQYTNQGKSNSQQRLKRLEKSLKELGSKILKENIPAKLSKIDNNALQNDRLYLYYLQNGKDMY TGDDLDIDRLSNYDIDHIIPQAFLKDNSIDNKVLVSSASNRGKSDDVPSLEVVKKRKTFWYQLLKSKLISQRKFDNLTKAERG GLSPEDKAGFIQRQLVETRQITKHVARLLDEKFNNKKDENNRAVRTVKIITLKSTLVSQFRKDFELYKVREINDFHHAHDAYL NAVVASALLKKYPKLEPEFVYGDYPKYNSFRERKSATEKVYFYSNIMNIFKKSISLADGRVIERPLIEVNEETGESVWNKESD LATVRRVLSYPQVNVVKKVEEQNHGLDRGKPKGLFNANLSSKPKPNSNENLVGAKEYLDPKKYGGYAGISNSFTVLVKGTIEK GAKKKITNVLEFQGISILDRINYRKDKLNFLLEKGYKDIELIIELPKYSLFELSDGSRRMLASILSTNNKRGEIHKGNQIFLS QKFVKLLYHAKRISNTINENHRKYVENHKKEFEELFYYILEFNENYVGAKKNGKLLNSAFQSWQNHSIDELCSSFIGPTGSER KGLFELTSRGSAADFEFLGVKIPRYRDYTPSSLLKDATLIHQSVTGLYETRIDLAKLGEG SEQ ID NO: 28 MKKQKFSDYYLGFDIGTNSVGWCVTDLDYNVLRFNKKDMWGSRLFDEAKTAAERRVQRNSRRRLKRRKWRLNLLEEIFSDEIM KIDSNFFRRLKESSLWLEDKNSKEKFTLFNDDNYKDYDFYKQYPTIFHLRDELIKNPEKKDIRLIYLALHSIFKSRGHFLFEG QNLKEIKNFETLYNNLISFLEDNGINKSIDKDNIEKLEKIICDSGKGLKDKEKEFKGIFNSDKQLVAIFKLSVGSSVSLNDLF DTDEYKKEEVEKEKISFREQIYEDDKPIYYSILGEKIELLDIAKSFYDFMVLNNILSDSNYISEAKVKLYEEHKKDLKNLKYI IRKYNKENYDKLFKDKNENNYPAYIGLNKEKDKKEVVEKSRLKIDDLIKVIKGYLPKPERIEEKDKTIFNEILNKIELKTILP KQRISDNGTLPYQIHEVELEKILENQSKYYDFLNYEENGVSTKDKLLKTFKFRIPYYVGPLNSYHKDKGGNSWIVRKEEGKIL PWNFEQKVDIEKSAEEFIKRMTNKCTYLNGEDVIPKDSFLYSEYIILNELNKVQVNDEFLNEENKRKIIDELFKENKKVSEKK FKEYLLVNQIANRTVELKGIKDSFNSNYVSYIKFKDIFGEKLNLDIYKEISEKSILWKCLYGDDKKIFEKKIKNEYGDILNKD EIKKINSFKFNTWGRLSEKLLTGIEFINLETGECYSSVMEALRRTNYNLMELLSSKFTLQESIDNENKEMNEVSYRDLIEESY VSPSLKRAILQTLKIYEEIKKITGRVPKKVFIEMARGGDESMKNKKIPARQEQLKKLYDSCGNDIANFSIDIKEMKNSLSSYD NNSLRQKKLYLYYLQFGKCMYTGREIDLDRLLQNNDTYDIDHIYPRSKVIKDDSFDNLVLVLKNENAEKSNEYPVKKEIQEKM KSFWRFLKEKNFISDEKYKRLTGKDDFELRGFMARQLVNVRQTTKEVGKILQQIEPEIKIVYSKAEIASSFREMFDFIKVREL NDTHHAKDAYLNIVAGNVYNTKFTEKPYRYLQEIKENYDVKKIYNYDIKNAWDKENSLEIVKKNMEKNTVNITRFIKEEKGEL FNLNPIKKGETSNEIISIKPKLYDGKDNKLNEKYGYYTSLKAAYFIYVEHEKKNKKVKTFERITRIDSTLIKNEKNLIKYLVS QKKLLNPKIIKKIYKEQTLIIDSYPYTFTGVDSNKKVELKNKKQLYLEKKYEQILKNALKFVEDNQGETEENYKFIYLKKRNN NEKNETIDAVKERYNIEFNEMYDKFLEKLSSKDYKNYINNKLYTNFLNSKEKFKKLKLWEKSLILREFLKIFNKNTYGKYEIK DSQTKEKLFSFPEDTGRIRLGQSSLGNNKELLEESVTGLFVKKIKL SEQ ID NO: 29 MKNYTIGLDIGVASVGWVCIDENYKILNYNNRHAFGVHEFESAESAAGRRLKRGMRRRYNRRKKRLQLLQSLFDSYITDSGFF SKTDSQHFWKNNNEFENRSLTEVLSSLRISSRKYPTIYHLRSDLIESNKKMDLRLVYLALHNLVKYRGHFLQEGNWSEAASAE GMDDQLLELVTRYAELENLSPLDLSESQWKAAETLLLNRNLTKTDQSKELTAMFGKEYEPFCKLVAGLGVSLHQLFPSSEQAL AYKETKTKVQLSNENVEEVMELLLEEESALLEAVQPFYQQVVLYELLKGETYVAKAKVSAFKQYQKDMASLKNLLDKTFGEKV YRSYFISDKNSQREYQKSHKVEVLCKLDQFNKEAKFAETFYKDLKKLLEDKSKTSIGTTEKDEMLRIIKAIDSNQFLQKQKGI QNAAIPHQNSLYEAEKILRNQQAHYPFITTEWIEKVKQILAFRIPYYIGPLVKDTTQSPFSWVERKGDAPITPWNFDEQIDKA ASAEAFISRMRKTCTYLKGQEVLPKSSLTYERFEVLNELNGIQLRTTGAESDFRHRLSYEMKCWIIDNVFKQYKTVSTKRLLQ ELKKSPYADELYDEHTGEIKEVFGTQKENAFATSLSGYISMKSILGAVVDDNPAMTEELIYWIAVFEDREILHLKIQEKYPSI TDVQRQKLALVKLPGWGRFSRLLIDGLPLDEQGQSVLDHMEQYSSVFMEVLKNKGFGLEKKIQKMNQHQVDGTKKIRYEDIEE LAGSPALKRGIWRSVKIVEELVSIFGEPANIVLEVAREDGEKKRTKSRKDQWEELTKTTLKNDPDLKSFIGEIKSQGDQRFNE QRFWLYVTQQGKCLYTGKALDIQNLSMYEVDHILPQNFVKDDSLDNLALVMPEANQRKNQVGQNKMPLEIIEANQQYAMRTLW ERLHELKLISSGKLGRLKKPSFDEVDKDKFIARQLVETRQIIKHVRDLLDERFSKSDIHLVKAGIVSKFRRFSEIPKIRDYNN KHHAMDALFAAALIQSILGKYGKNFLAFDLSKKDRQKQWRSVKGSNKEFFLFKNFGNLRLQSPVTGEEVSGVEYMKHVYFELP WQTTKMTQTGDGMFYKESIFSPKVKQAKYVSPKTEKFVHDEVKNHSICLVEFTFMKKEKEVQETKFIDLKVIEHHQFLKEPES QLAKFLAEKETNSPIIHARIIRTIPKYQKIWIEHFPYYFISTRELHNARQFEISYELMEKVKQLSERSSVEELKIVFGLLIDQ MNDNYPIYTKSSIQDRVQKFVDTQLYDFKSFEIGFEELKKAVAANAQRSDTFGSRISKKPKPEEVAIGYESITGLKYRKPRSV VGTKR SEQ ID NO: 30 MKKEIKDYFLGLDVGTGSVGWAVTDTDYKLLKANRKDLWGMRCFETAETAEVRRLHRGARRRIERRKKRIKLLQELFSQEIAK TDEGFFQRMKESPFYAEDKTILQENTLFNDKDFADKTYHKAYPTINHLIKAWIENKVKPDPRLLYLACHNIIKKRGHFLFEGD FDSENQFDTSIQALFEYLREDMEVDIDADSQKVKEILKDSSLKNSEKQSRLNKILGLKPSDKQKKAITNLISGNKINFADLYD NPDLKDAEKNSISFSKDDFDALSDDLASILGDSFELLLKAKAVYNCSVLSKVIGDEQYLSFAKVKIYEKHKTDLTKLKNVIKK HFPKDYKKVFGYNKNEKNNNNYSGYVGVCKTKSKKLIINNSVNQEDFYKFLKTILSAKSEIKEVNDILTEIETGTFLPKQISK SNAEIPYQLRKMELEKILSNAEKHFSFLKQKDEKGLSHSEKIIMLLTFKIPYYIGPINDNHKKFFPDRCWVVKKEKSPSGKTT PWNFFDHIDKEKTAEAFITSRTNFCTYLVGESVLPKSSLLYSEYTVLNEINNLQIIIDGKNICDIKLKQKIYEDLFKKYKKIT QKQISTFIKHEGICNKTDEVIILGIDKECTSSLKSYIELKNIFGKQVDEISTKNMLEEIIRWATIYDEGEGKTILKTKIKAEY GKYCSDEQIKKILNLKFSGWGRLSRKFLETVTSEMPGFSEPVNIITAMRETQNNLMELLSSEFTFTENIKKINSGFEDAEKQF SYDGLVKPLFLSPSVKKMLWQTLKLVKEISHITQAPPKKIFIEMAKGAELEPARTKTRLKILQDLYNNCKNDADAFSSEIKDL SGKIENEDNLRLRSDKLYLYYTQLGKCMYCGKPIEIGHVFDTSNYDIDHIYPQSKIKDDSISNRVLVCSSCNKNKEDKYPLKS EIQSKQRGFWNFLQRNNFISLEKLNRLTRATPISDDETAKFIARQLVETRQATKVAAKVLEKMFPETKIVYSKAETVSMFRNK FDIVKCREINDFHHAHDAYLNIVVGNVYNTKFTNNPWNFIKEKRDNPKIADTYNYYKVFDYDVKRNNITAWEKGKTIITVKDM LKRNTPIYTRQAACKKGELFNQTIMKKGLGQHPLKKEGPFSNISKYGGYNKVSAAYYTLIEYEEKGNKIRSLETIPLYLVKDI QKDQDVLKSYLTDLLGKKEFKILVPKIKINSLLKINGFPCHITGKTNDSFLLRPAVQFCCSNNEVLYFKKIIRFSEIRSQREK IGKTISPYEDLSFRSYIKENLWKKTKNDEIGEKEFYDLLQKKNLEIYDMLLTKHKDTIYKKRPNSATIDILVKGKEKFKSLII ENQFEVILEILKLFSATRNVSDLQHIGGSKYSGVAKIGNKISSLDNCILIYQSITGIFEKRIDLLKV SEQ ID NO: 31 MEGQMKNNGNNLQQGNYYLGLDVGTSSVGWAVTDTDYNVLKFRGKSMWGARLFDEASTAEERRTHRGNRRRLARRKYRLLLLE QLFEKEIRKIDDNFFVRLHESNLWADDKSKPSKFLLFNDTNFTDKDYLKKYPTIYHLRSDLIHNSTEHDIRLVFLALHHLIKY RGHFIYDNSANGDVKTLDEAVSDFEEYLNENDIEFNIENKKEFINVLSDKHLTKKEKKISLKKLYGDITDSENINISVLIEML SGSSISLSNLFKDIEFDGKQNLSLDSDIEETLNDVVDILGDNIDLLIHAKEVYDIAVLTSSLGKHKYLCDAKVELFEKNKKDL MILKKYIKKNHPEDYKKIFSSPTEKKNYAAYSQTNSKNVCSQEEFCLFIKPYIRDMVKSENEDEVRIAKEVEDKSFLTKLKGT NNSVVPYQIHERELNQILKNIVAYLPFMNDEQEDISVVDKIKLIFKFKIPYYVGPLNTKSTRSWVYRSDEKIYPWNFSNVIDL DKTAHEFMNRLIGRCTYTNDPVLPMDSLLYSKYNVLNEINPIKVNGKAIPVEVKQAIYTDLFENSKKKVTRKSIYIYLLKNGY IEKEDIVSGIDIEIKSKLKSHHDFTQIVQENKCTPEEIERIIKGILVYSDDKSMLRRWLKNNIKGLSENDVKYLAKLNYKEWG RLSKTLLTDIYTINPEDGEACSILDIMWNTNATLMEILSNEKYQFKQNIENYKAENYDEKQNLHEELDDMYISPAARRSIWQA LRIVDEIVDIKKSAPKKIFIEMAREKKSAMKKKRTESRKDTLLELYKSCKSQADGFYDEELFEKLSNESNSRLRRDQLYLYYT QMGRSMYTGKRIDFDKLINDKNTYDIDHIYPRSKIKDDSITNRVLVEKDINGEKTDIYPISEDIRQKMQPFWKILKEKGLINE EKYKRLTRNYELTDEELSSFVARQLVETQQSTKALATLLKKEYPSAKIVYSKAGNVSEFRNRKDKELPKFREINDLHHAKDAY LNIVVGNVYDTKFTEKFFNNIRNENYSLKRVFDFSVPGAWDAKGSTFNTIKKYMAKNNPIIAFAPYEVKGELFDQQIVPKGKG QFPIKQGKDIEKYGGYNKLSSAFLFAVEYKGKKARERSLETVYIKDVELYLQDPIKYCESVLGLKEPQIIKPKILMGSLFSIN NKKLVVTGRSGKQYVCHHIYQLSINDEDSQYLKNIAKYLQEEPDGNIERQNILNITSVNNIKLFDVLCTKFNSNTYEIILNSL KNDVNEGREKFSELDILEQCNILLQLLKAFKCNRESSNLEKLNNKKQAGVIVIPHLFTKCSVFKVIHQSITGLFEKEMDLLK SEQ ID NO: 32 MGRKPYILSLDIGTGSVGYACMDKGFNVLKYHDKDALGVYLFDGALTAQERRQFRTSRRRKNRRIKRLGLLQELLAPLVQNPN FYQFQRQFAWKNDNMDFKNKSLSEVLSFLGYESKKYPTIYHLQEALLLKDEKFDPELIYMALYHLVKYRGHFLFDHLKIENLT NNDNMHDFVELIETYENLNNIKLNLDYEKTKVIYEILKDNEMTKNDRAKRVKNMEKKLEQFSIMLLGLKFNEGKLFNHADNAE ELKGANQSHTFADNYEENLTPFLTVEQSEFIERANKIYLSLTLQDILKGKKSMAMSKVAAYDKFRNELKQVKDIVYKADSTRT QFKKIFVSSKKSLKQYDATPNDQTFSSLCLFDQYLIRPKKQYSLLIKELKKIIPQDSELYFEAENDTLLKVLNTTDNASIPMQ INLYEAETILRNQQKYHAEITDEMIEKVLSLIQFRIPYYVGPLVNDHTASKFGWMERKSNESIKPWNFDEVVDRSKSATQFIR RMTNKCSYLINEDVLPKNSLLYQEMEVLNELNATQIRLQTDPKNRKYRMMPQIKLFAVEHIFKKYKTVSHSKFLEIMLNSNHR ENFMNHGEKLSIFGTQDDKKFASKLSSYQDMTKIFGDIEGKRAQIEEIIQWITIFEDKKILVQKLKECYPELTSKQINQLKKL NYSGWGRLSEKLLTHAYQGHSIIELLRHSDENFMEILTNDVYGFQNFIKEENQVQSNKIQHQDIANLTTSPALKKGIWSTIKL VRELTSIFGEPEKIIMEFATEDQQKGKKQKSRKQLWDDNIKKNKLKSVDEYKYIIDVANKLNNEQLQQEKLWLYLSQNGKCMY SGQSIDLDALLSPNATKHYEVDHIFPRSFIKDDSIDNKVLVIKKMNQTKGDQVPLQFIQQPYERIAYWKSLNKAGLISDSKLH KLMKPEFTAMDKEGFIQRQLVETRQISVHVRDFLKEEYPNTKVIPMKAKMVSEFRKKFDIPKIRQMNDAHHAIDAYLNGVVYH GAQLAYPNVDLFDFNFKWEKVREKWKALGEFNTKQKSRELFFFKKLEKMEVSQGERLISKIKLDMNHFKINYSRKLANIPQQF YNQTAVSPKTAELKYESNKSNEVVYKGLTPYQTYVVAIKSVNKKGKEKMEYQMIDHYVFDFYKFQNGNEKELALYLAQRENKD EVLDAQIVYSLNKGDLLYINNHPCYFVSRKEVINAKQFELTVEQQLSLYNVMNNKETNVEKLLIEYDFIAEKVINEYHHYLNS KLKEKRVRTFFSESNQTHEDFIKALDELFKVVTASATRSDKIGSRKNSMTHRAFLGKGKDVKIAYTSISGLKTTKPKSLFKLA ESRNEL SEQ ID NO: 33 MAKILGLDLGTNSIGWAVVERENIDFSLIDKGVRIFSEGVKSEKGIESSRAAERTGYRSARKIKYRRKLRKYETLKVLSLNRM CPLSIEEVEEWKKSGFKDYPLNPEFLKWLSTDEESNVNPYFFRDRASKHKVSLFELGRAFYHIAQRRGFLSNRLDQSAEGILE EHCPKIEAIVEDLISIDEISTNITDYFFETGILDSNEKNGYAKDLDEGDKKLVSLYKSLLAILKKNESDFENCKSEIIERLNK KDVLGKVKGKIKDISQAMLDGNYKTLGQYFYSLYSKEKIRNQYTSREEHYLSEFITICKVQGIDQINEEEKINEKKFDGLAKD LYKAIFFQRPLKSQKGLIGKCSFEKSKSRCAISHPDFEEYRMWTYLNTIKIGTQSDKKLRFLTQDEKLKLVPKFYRKNDFNFD VLAKELIEKGSSFGFYKSSKKNDFFYWFNYKPTDTVAACQVAASLKNAIGEDWKTKSFKYQTINSNKEQVSRTVDYKDLWHLL TVATSDVYLYEFAIDKLGLDEKNAKAFSKTKLKKDFASLSLSAINKILPYLKEGLLYSHAVFVANIENIVDENIWKDEKQRDY IKTQISEIIENYTLEKSRFEIINGLLKEYKSENEDGKRVYYSKEAEQSFENDLKKKLVLFYKSNEIENKEQQETIFNELLPIF IQQLKDYEFIKIQRLDQKVLIFLKGKNETGQIFCTEEKGTAEEKEKKIKNRLKKLYHPSDIEKFKKKIIKDEFGNEKIVLGSP LTPSIKNPMAMRALHQLRKVLNALILEGQIDEKTIIHIEMARELNDANKRKGIQDYQNDNKKFREDAIKEIKKLYFEDCKKEV EPTEDDILRYQLWMEQNRSEIYEEGKNISICDIIGSNPAYDIEHTIPRSRSQDNSQMNKTLCSQRFNREVKKQSMPIELNNHL EILPRIAHWKEEADNLTREIEIISRSIKAAATKEIKDKKIRRRHYLTLKRDYLQGKYDRFIWEEPKVGFKNSQIPDTGIITKY AQAYLKSYFKKVESVKGGMVAEFRKIWGIQESFIDENGMKHYKVKDRSKHTHHTIDAITIACMTKEKYDVLAHAWTLEDQQNK KEARSIIEASKPWKTFKEDLLKIEEEILVSHYTPDNVKKQAKKIVRVRGKKQFVAEVERDVNGKAVPKKAASGKTIYKLDGEG KKLPRLQQGDTIRGSLHQDSIYGAIKNPLNTDEIKYVIRKDLESIKGSDVESIVDEVVKEKIKEAIANKVLLLSSNAQQKNKL VGTVWMNEEKRIAINKVRIYANSVKNPLHIKEHSLLSKSKHVHKQKVYGQNDENYAMAIYELDGKRDFELINIFNLAKLIKQG QGFYPLHKKKEIKGKIVFVPIEKRNKRDVVLKRGQQVVFYDKEVENPKDISEIVDFKGRIYIIEGLSIQRIVRPSGKVDEYGV IMLRYFKEARKADDIKQDNFKPDGVFKLGENKPTRKMNHQFTAFVEGIDFKVLPSGKFEKI SEQ ID NO: 34 MEFKKVLGLDIGTNSIGCALLSLPKSIQDYGKGGRLEWLTSRVIPLDADYMKAFIDGKNGLPQVITPAGKRRQKRGSRRLKHR YKLRRSRLIRVFKTLNWLPEDFPLDNPKRIKETISTEGKFSFRISDYVPISDESYREFYREFGYPENEIEQVIEEINFRRKTK GKNKNPMIKLLPEDWVVYYLRKKALIKPTTKEELIRIIYLFNQRRGFKSSRKDLTETAILDYDEFAKRLAEKEKYSAENYETK FVSITKVKEVVELKTDGRKGKKRFKVILEDSRIEPYEIERKEKPDWEGKEYTFLVTQKLEKGKFKQNKPDLPKEEDWALCTTA LDNRMGSKHPGEFFFDELLKAFKEKRGYKIRQYPVNRWRYKKELEFIWTKQCQLNPELNNLNINKEILRKLATVLYPSQSKFF GPKIKEFENSDVLHIISEDIIYYQRDLKSQKSLISECRYEKRKGIDGEIYGLKCIPKSSPLYQEFRIWQDIHNIKVIRKESEV NGKKKINIDETQLYINENIKEKLFELFNSKDSLSEKDILELISLNIINSGIKISKKEEETTHRINLFANRKELKGNETKSRYR KVFKKLGFDGEYILNHPSKLNRLWHSDYSNDYADKEKTEKSILSSLGWKNRNGKWEKSKNYDVFNLPLEVAKAIANLPPLKKE YGSYSALAIRKMLVVMRDGKYWQHPDQIAKDQENTSLMLFDKNLIQLTNNQRKVLNKYLLTLAEVQKRSTLIKQKLNEIEHNP YKLELVSDQDLEKQVLKSFLEKKNESDYLKGLKTYQAGYLIYGKHSEKDVPIVNSPDELGEYIRKKLPNNSLRNPIVEQVIRE TIFIVRDVWKSFGIIDEIHIELGRELKNNSEERKKTSESQEKNFQEKERARKLLKELLNSSNFEHYDENGNKIFSSFTVNPNP DSPLDIEKFRIWKNQSGLTDEELNKKLKDEKIPTEIEVKKYILWLTQKCRSPYTGKIIPLSKLFDSNVYEIEHIIPRSKMKND STNNLVICELGVNKAKGDRLAANFISESNGKCKFGEVEYTLLKYGDYLQYCKDTFKYQKAKYKNLLATEPPEDFIERQINDTR YIGRKLAELLTPVVKDSKNIIFTIGSITSELKITWGLNGVWKDILRPRFKRLESIINKKLIFQDEDDPNKYHFDLSINPQLDK EGLKRLDHRHHALDATIIAATTREHVRYLNSLNAADNDEEKREYFLSLCNHKIRDFKLPWENFTSEVKSKLLSCVVSYKESKP ILSDPFNKYLKWEYKNGKWQKVFAIQIKNDRWKAVRRSMFKEPIGTVWIKKIKEVSLKEAIKIQAIWEEVKNDPVRKKKEKYI YDDYAQKVIAKIVQELGLSSSMRKQDDEKLNKFINEAKVSAGVNKNLNTTNKTIYNLEGRFYEKIKVAEYVLYKAKRMPLNKK EYIEKLSLQKMFNDLPNFILEKSILDNYPEILKELESDNKYIIEPHKKNNPVNRLLLEHILEYHNNPKEAFSTEGLEKLNKKA INKIGKPIKYITRLDGDINEEEIFRGAVFETDKGSNVYFVMYENNQTKDREFLKPNPSISVLKAIEHKNKIDFFAPNRLGFSR IILSPGDLVYVPTNDQYVLIKDNSSNETIINWDDNEFISNRIYQVKKFTGNSCYFLKNDIASLILSYSASNGVGEFGSQNISE YSVDDPPIRIKDVCIKIRVDRLGNVRPL SEQ ID NO: 35 MKHILGLDLGTNSIGWALIERNIEEKYGKIIGMGSRIVPMGAELSKFEQGQAQTKNADRRTNRGARRLNKRYKQRRNKLIYIL QKLDMLPSQIKLKEDFSDPNKIDKITILPISKKQEQLTAFDLVSLRVKALTEKVGLEDLGKIIYKYNQLRGYAGGSLEPEKED IFDEEQSKDKKNKSFIAFSKIVFLGEPQEEIFKNKKLNRRAIIVETEEGNFEGSTFLENIKVGDSLELLINISASKSGDTITI KLPNKTNWRKKMENIENQLKEKSKEMGREFYISEFLLELLKENRWAKIRNNTILRARYESEFEAIWNEQVKHYPFLENLDKKT LIEIVSFIFPGEKESQKKYRELGLEKGLKYIIKNQVVFYQRELKDQSHLISDCRYEPNEKAIAKSHPVFQEYKVWEQINKLIV NTKIEAGTNRKGEKKYKYIDRPIPTALKEWIFEELQNKKEITFSAIFKKLKAEFDLREGIDFLNGMSPKDKLKGNETKLQLQK SLGELWDVLGLDSINRQIELWNILYNEKGNEYDLTSDRTSKVLEFINKYGNNIVDDNAEETAIRISKIKFARAYSSLSLKAVE RILPLVRAGKYFNNDFSQQLQSKILKLLNENVEDPFAKAAQTYLDNNQSVLSEGGVGNSIATILVYDKHTAKEYSHDELYKSY KEINLLKQGDLRNPLVEQIINEALVLIRDIWKNYGIKPNEIRVELARDLKNSAKERATIHKRNKDNQTINNKIKETLVKNKKE LSLANIEKVKLWEAQRHLSPYTGQPIPLSDLFDKEKYDVDHIIPISRYFDDSFTNKVISEKSVNQEKANRTAMEYFEVGSLKY SIFTKEQFIAHVNEYFSGVKRKNLLATSIPEDPVQRQIKDTQYIAIRVKEELNKIVGNENVKTTTGSITDYLRNHWGLTDKFK LLLKERYEALLESEKFLEAEYDNYKKDFDSRKKEYEEKEVLFEEQELTREEFIKEYKENYIRYKKNKLIIKGWSKRIDHRHHA IDALIVACTEPAHIKRLNDLNKVLQDWLVEHKSEFMPNFEGSNSELLEEILSLPENERTEIFTQIEKFRAIEMPWKGFPEQVE QKLKEIIISHKPKDKLLLQYNKAGDRQIKLRGQLHEGTLYGISQGKEAYRIPLTKFGGSKFATEKNIQKIVSPFLSGFIANHL KEYNNKKEEAFSAEGIMDLNNKLAQYRNEKGELKPHTPISTVKIYYKDPSKNKKKKDEEDLSLQKLDREKAFNEKLYVKTGDN YLFAVLEGEIKTKKTSQIKRLYDIISFFDATNFLKEEFRNAPDKKTFDKDLLFRQYFEERNKAKLLFTLKQGDFVYLPNENEE VILDKESPLYNQYWGDLKERGKNIYVVQKFSKKQIYFIKHTIADIIKKDVEFGSQNCYETVEGRSIKENCFKLEIDRLGNIVK VIKR SEQ ID NO: 36 MHVEIDFPHFSRGDSHLAMNKNEILRGSSVLYRLGLDLGSNSLGWFVTHLEKRGDRHEPVALGPGGVRIFPDGRDPQSGTSNA VDRRMARGARKRRDRFVERRKELIAALIKYNLLPDDARERRALEVLDPYALRKTALTDTLPAHHVGRALFHLNQRRGFQSNRK TDSKQSEDGAIKQAASRLATDKGNETLGVFFADMHLRKSYEDRQTAIRAELVRLGKDHLTGNARKKIWAKVRKRLFGDEVLPR ADAPHGVRARATITGTKASYDYYPTRDMLRDEFNAIWAGQSAHHATITDEARTEIEHIIFYQRPLKPAIVGKCTLDPATRPFK EDPEGYRAPWSHPLAQRFRILSEARNLEIRDTGKGSRRLTKEQSDLVVAALLANREVKFDKLRTLLKLPAEARFNLESDRRAA LDGDQTAARLSDKKGFNKAWRGFPPERQIAIVARLEETEDENELIAWLEKECALDGAAAARVANTTLPDGHCRLGLRAIKKIV PIMQDGLDEDGVAGAGYHIAAKRAGYDHAKLPTGEQLGRLPYYGQWLQDAVVGSGDARDQKEKQYGQFPNPTVHIGLGQLRRV VNDLIDKYGPPTEISIEFTRALKLSEQQKAERQREQRRNQDKNKARAEELAKFGRPANPRNLLKMRLWEELAHDPLDRKCVYT GEQISIERLLSDEVDIDHILPVAMTLDDSPANKIICMRYANRHKRKQTPSEAFGSSPTLQGHRYNWDDIAARATGLPRNKRWR FDANAREEFDKRGGFLARQLNETGWLARLAKQYLGAVTDPNQIWVVPGRLTSMLRGKWGLNGLLPSDNYAGVQDKAEEFLAST DDMEFSGVKNRADHRHHAIDGLVTALTDRSLLWKMANAYDEEHEKFVIEPPWPTMRDDLKAALEKMVVSHKPDHGIEGKLHED SAYGFVKPLDATGLKEEEAGNLVYRKAIESLNENEVDRIRDIQLRTIVRDHVNVEKTKGVALADALRQLQAPSDDYPQFKHGL RHVRILKKEKGDYLVPIANRASGVAYKAYSAGENFCVEVFETAGGKWDGEAVRRFDANKKNAGPKIAHAPQWRDANEGAKLVM RIHKGDLIRLDHEGRARIMVVHRLDAAAGRFKLADHNETGNLDKRHATNNDIDPFRWLMASYNTLKKLAAVPVRVDELGRVWR VMPN SEQ ID NO: 37 METTLGIDLGTNSIGLALVDQEEHQILYSGVRIFPEGINKDTIGLGEKEESRNATRRAKRQMRRQYFRKKLRKAKLLELLIAY DMCPLKPEDVRRWKNWDKQQKSTVRQFPDTPAFREWLKQNPYELRKQAVTEDVTRPELGRILYQMIQRRGFLSSRKGKEEGKI FTGKDRMVGIDETRKNLQKQTLGAYLYDIAPKNGEKYRFRTERVRARYTLRDMYIREFEIIWQRQAGHLGLAHEQATRKKNIF LEGSATNVRNSKLITHLQAKYGRGHVLIEDTRITVTFQLPLKEVLGGKIEIEEEQLKFKSNESVLFWQRPLRSQKSLLSKCVF EGRNFYDPVHQKWIIAGPTPAPLSHPEFEEFRAYQFINNIIYGKNEHLTAIQREAVFELMCTESKDFNFEKIPKHLKLFEKFN FDDTTKVPACTTISQLRKLFPHPVWEEKREEIWHCFYFYDDNTLLFEKLQKDYALQTNDLEKIKKIRLSESYGNVSLKAIRRI NPYLKKGYAYSTAVLLGGIRNSFGKRFEYFKEYEPEIEKAVCRILKEKNAEGEVIRKIKDYLVHNRFGFAKNDRAFQKLYHHS QAITTQAQKERLPETGNLRNPIVQQGLNELRRTVNKLLATCREKYGPSFKFDHIHVEMGRELRSSKTEREKQSRQIRENEKKN EAAKVKLAEYGLKAYRDNIQKYLLYKEIEEKGGTVCCPYTGKTLNISHTLGSDNSVQIEHIIPYSISLDDSLANKTLCDATFN REKGELTPYDFYQKDPSPEKWGASSWEEIEDRAFRLLPYAKAQRFIRRKPQESNEFISRQLNDTRYISKKAVEYLSAICSDVK AFPGQLTAELRHLWGLNNILQSAPDITFPLPVSATENHREYYVITNEQNEVIRLFPKQGETPRTEKGELLLTGEVERKVFRCK GMQEFQTDVSDGKYWRRIKLSSSVTWSPLFAPKPISADGQIVLKGRIEKGVFVCNQLKQKLKTGLPDGSYWISLPVISQTFKE GESVNNSKLTSQQVQLFGRVREGIFRCHNYQCPASGADGNFWCTLDTDTAQPAFTPIKNAPPGVGGGQIILTGDVDDKGIFHA DDDLHYELPASLPKGKYYGIFTVESCDPTLIPIELSAPKTSKGENLIEGNIWVDEHTGEVRFDPKKNREDQRHHAIDAIVIAL SSQSLFQRLSTYNARRENKKRGLDSTEHFPSPWPGFAQDVRQSVVPLLVSYKQNPKTLCKISKTLYKDGKKIHSCGNAVRGQL HKETVYGQRTAPGATEKSYHIRKDIRELKTSKHIGKVVDITIRQMLLKHLQENYHIDITQEFNIPSNAFFKEGVYRIFLPNKH GEPVPIKKIRMKEELGNAERLKDNINQYVNPRNNHHVMIYQDADGNLKEEIVSFWSVIERQNQGQPIYQLPREGRNIVSILQI NDTFLIGLKEEEPEVYRNDLSTLSKHLYRVQKLSGMYYTFRHHLASTLNNEREEFRIQSLEAWKRANPVKVQIDEIGRITFLN GPLC SEQ ID NO: 38 MESSQILSPIGIDLGGKFTGVCLSHLEAFAELPNHANTKYSVILIDHNNFQLSQAQRRATRHRVRNKKRNQFVKRVALQLFQH ILSRDLNAKEETALCHYLNNRGYTYVDTDLDEYIKDETTINLLKELLPSESEHNFIDWFLQKMQSSEFRKILVSKVEEKKDDK ELKNAVKNIKNFITGFEKNSVEGHRHRKVYFENIKSDITKDNQLDSIKKKIPSVCLSNLLGHLSNLQWKNLHRYLAKNPKQFD EQTFGNEFLRMLKNFRHLKGSQESLAVRNLIQQLEQSQDYISILEKTPPEITIPPYEARTNTGMEKDQSLLLNPEKLNNLYPN WRNLIPGIIDAHPFLEKDLEHTKLRDRKRIISPSKQDEKRDSYILQRYLDLNKKIDKFKIKKQLSFLGQGKQLPANLIETQKE METHFNSSLVSVLIQIASAYNKEREDAAQGIWFDNAFSLCELSNINPPRKQKILPLLVGAILSEDFINNKDKWAKFKIFWNTH KIGRTSLKSKCKEIEEARKNSGNAFKIDYEEALNHPEHSNNKALIKIIQTIPDIIQAIQSHLGHNDSQALIYHNPFSLSQLYT ILETKRDGFHKNCVAVTCENYWRSQKTEIDPEISYASRLPADSVRPFDGVLARMMQRLAYEIAMAKWEQIKHIPDNSSLLIPI YLEQNRFEFEESFKKIKGSSSDKTLEQAIEKQNIQWEEKFQRIINASMNICPYKGASIGGQGEIDHIYPRSLSKKHFGVIFNS EVNLIYCSSQGNREKKEEHYLLEHLSPLYLKHQFGTDNVSDIKNFISQNVANIKKYISFHLLTPEQQKAARHALFLDYDDEAF KTITKFLMSQQKARVNGTQKFLGKQIMEFLSTLADSKQLQLEFSIKQITAEEVHDHRELLSKQEPKLVKSRQQSFPSHAIDAT LTMSIGLKEFPQFSQELDNSWFINHLMPDEVHLNPVRSKEKYNKPNISSTPLFKDSLYAERFIPVWVKGETFAIGFSEKDLFE IKPSNKEKLFTLLKTYSTKNPGESLQELQAKSKAKWLYFPINKTLALEFLHHYFHKEIVTPDDTTVCHFINSLRYYTKKESIT VKILKEPMPVLSVKFESSKKNVLGSFKHTIALPATKDWERLFNHPNFLALKANPAPNPKEFNEFIRKYFLSDNNPNSDIPNNG HNIKPQKHKAVRKVFSLPVIPGNAGTMMRIRRKDNKGQPLYQLQTIDDTPSMGIQINEDRLVKQEVLMDAYKTRNLSTIDGIN NSEGQAYATFDNWLTLPVSTFKPEIIKLEMKPHSKTRRYIRITQSLADFIKTIDEALMIKPSDSIDDPLNMPNEIVCKNKLFG NELKPRDGKMKIVSTGKIVTYEFESDSTPQWIQTLYVTQLKKQP SEQ ID NO: 39 MKKIVGLDLGTNSIGWALINAYINKEHLYGIEACGSRIIPMDAAILGNFDKGNSISQTADRTSYRGIRRLRERHLLRRERLHR ILDLLGFLPKHYSDSLNRYGKFLNDIECKLPWVKDETGSYKFIFQESFKEMLANFTEHHPILIANNKKVPYDWTIYYLRKKAL TQKISKEELAWILLNFNQKRGYYQLRGEEEETPNKLVEYYSLKVEKVEDSGERKGKDTWYNVHLENGMIYRRTSNIPLDWEGK TKEFIVTTDLEADGSPKKDKEGNIKRSFRAPKDDDWTLIKKKTEADIDKIKMTVGAYIYDTLLQKPDQKIRGKLVRTIERKYY KNELYQILKTQSEFHEELRDKQLYIACLNELYPNNEPRRNSISTRDFCHLFIEDIIFYQRPLKSKKSLIDNCPYEENRYIDKE SGEIKHASIKCIAKSHPLYQEFRLWQFIVNLRIYRKETDVDVTQELLPTEADYVTLFEWLNEKKEIDQKAFFKYPPFGFKKTT SNYRWNYVEDKPYPCNETHAQIIARLGKAHIPKAFLSKEKEETLWHILYSIEDKQEIEKALHSFANKNNLSEEFIEQFKNFPP FKKEYGSYSAKAIKKLLPLMRMGKYWSIENIDNGTRIRINKIIDGEYDENIRERVRQKAINLTDITHFRALPLWLACYLVYDR HSEVKDIVKWKTPKDIDLYLKSFKQHSLRNPIVEQVITETLRTVRDIWQQVGHIDEIHIELGREMKNPADKRARMSQQMIKNE NTNLRIKALLTEFLNPEFGIENVRPYSPSQQDLLRIYEEGVLNSILELPEDIGIILGKFNQTDTLKRPTRSEILRYKLWLEQK YRSPYTGEMIPLSKLFTPAYEIEHIIPQSRYFDDSLSNKVICESEINKLKDRSLGYEFIKNHHGEKVELAFDKPVEVLSVEAY EKLVHESYSHNRSKMKKLLMEDIPDQFIERQLNDSRYISKVVKSLLSNIVREENEQEAISKNVIPCTGGITDRLKKDWGINDV WNKIVLPRFIRLNELTESTRFTSINTNNTMIPSMPLELQKGFNKKRIDHRHHAMDAIIIACANRNIVNYLNNVSASKNTKITR RDLQTLLCHKDKTDNNGNYKWVIDKPWETFTQDTLTALQKITVSFKQNLRVINKTTNHYQHYENGKKIVSNQSKGDSWAIRKS MHKETVHGEVNLRMIKTVSFNEALKKPQAIVEMDLKKKILAMLELGYDTKRIKNYFEENKDTWQDINPSKIKVYYFTKETKDR YFAVRKPIDTSFDKKKIKESITDTGIQQIMLRHLETKDNDPTLAFSPDGIDEMNRNILILNKGKKHQPIYKVRVYEKAEKFTV GQKGNKRTKFVEAAKGTNLFFAIYETEEIDKDTKKVIRKRSYSTIPLNVVIERQKQGLSSAPEDENGNLPKYILSPNDLVYVP TQEEINKGEVVMPIDRDRIYKMVDSSGITANFIPASTANLIFALPKATAEIYCNGENCIQNEYGIGSPQSKNQKAITGEMVKE ICFPIKVDRLGNIIQVGSCILTN SEQ ID NO: 40 MSRSLTFSFDIGYASIGWAVIASASHDDADPSVCGCGTVLFPKDDCQAFKRREYRRLRRNIRSRRVRIERIGRLLVQAQIITP EMKETSGHPAPFYLASEALKGHRTLAPIELWHVLRWYAHNRGYDNNASWSNSLSEDGGNGEDTERVKHAQDLMDKHGTATMAE TICRELKLEEGKADAPMEVSTPAYKNLNTAFPRLIVEKEVRRILELSAPLIPGLTAEIIELIAQHHPLTTEQRGVLLQHGIKL ARRYRGSLLFGQLIPRFDNRIISRCPVTWAQVYEAELKKGNSEQSARERAEKLSKVPTANCPEFYEYRMARILCNIRADGEPL SAEIRRELMNQARQEGKLTKASLEKAISSRLGKETETNVSNYFTLHPDSEEALYLNPAVEVLQRSGIGQILSPSVYRIAANRL RRGKSVTPNYLLNLLKSRGESGEALEKKIEKESKKKEADYADTPLKPKYATGRAPYARTVLKKVVEEILDGEDPTRPARGEAH PDGELKAHDGCLYCLLDTDSSVNQHQKERRLDTMTNNHLVRHRMLILDRLLKDLIQDFADGQKDRISRVCVEVGKELTTFSAM DSKKIQRELTLRQKSHTDAVNRLKRKLPGKALSANLIRKCRIAMDMNWTCPFTGATYGDHELENLELEHIVPHSFRQSNALSS LVLTWPGVNRMKGQRTGYDFVEQEQENPVPDKPNLHICSLNNYRELVEKLDDKKGHEDDRRRKKKRKALLMVRGLSHKHQSQN HEAMKEIGMTEGMMTQSSHLMKLACKSIKTSLPDAHIDMIPGAVTAEVRKAWDVFGVFKELCPEAADPDSGKILKENLRSLTH LHHALDACVLGLIPYIIPAHHNGLLRRVLAMRRIPEKLIPQVRPVANQRHYVLNDDGRMMLRDLSASLKENIREQLMEQRVIQ HVPADMGGALLKETMQRVLSVDGSGEDAMVSLSKKKDGKKEKNQVKASKLVGVFPEGPSKLKALKAAIEIDGNYGVALDPKPV VIRHIKVFKRIMALKEQNGGKPVRILKKGMLIHLTSSKDPKHAGVWRIESIQDSKGGVKLDLQRAHCAVPKNKTHECNWREVD LISLLKKYQMKRYPTSYTGTPR SEQ ID NO: 41 MTQKVLGLDLGTNSIGSAVRNLDLSDDLQWQLEFFSSDIFRSSVNKESNGREYSLAAQRSAHRRSRGLNEVRRRRLWATLNLL IKHGFCPMSSESLMRWCTYDKRKGLFREYPIDDKDFNAWILLDFNGDGRPDYSSPYQLRRELVTRQFDFEQPIERYKLGRALY HIAQHRGFKSSKGETLSQQETNSKPSSTDEIPDVAGAMKASEEKLSKGLSTYMKEHNLLTVGAAFAQLEDEGVRVRNNNDYRA IRSQFQHEIETIFKFQQGLSVESELYERLISEKKNVGTIFYKRPLRSQRGNVGKCTLERSKPRCAIGHPLFEKFRAWTLINNI KVRMSVDTLDEQLPMKLRLDLYNECFLAFVRTEFKFEDIRKYLEKRLGIHFSYNDKTINYKDSTSVAGCPITARFRKMLGEEW ESFRVEGQKERQAHSKNNISFHRVSYSIEDIWHFCYDAEEPEAVLAFAQETLRLERKKAEELVRIWSAMPQGYAMLSQKAIRN INKILMLGLKYSDAVILAKVPELVDVSDEELLSIAKDYYLVEAQVNYDKRINSIVNGLIAKYKSVSEEYRFADHNYEYLLDES DEKDIIRQIENSLGARRWSLMDANEQTDILQKVRDRYQDFFRSHERKFVESPKLGESFENYLTKKFPMVEREQWKKLYHPSQI TIYRPVSVGKDRSVLRLGNPDIGAIKNPTVLRVLNTLRRRVNQLLDDGVISPDETRVVVETARELNDANRKWALDTYNRIRHD ENEKIKKILEEFYPKRDGISTDDIDKARYVIDQREVDYFTGSKTYNKDIKKYKFWLEQGGQCMYTGRTINLSNLFDPNAFDIE HTIPESLSFDSSDMNLTLCDAHYNRFIKKNHIPTDMPNYDKAITIDGKEYPAITSQLQRWVERVERLNRNVEYWKGQARRAQN KDRKDQCMREMHLWKMELEYWKKKLERFTVIEVIDGFKNSQLVDTRVITRHAVLYLKSIFPHVDVQRGDVTAKFRKILGIQSV DEKKDRSLHSHHAIDATTLTIIPVSAKRDRMLELFAKIEEINKMLSFSGSEDRTGLIQELEGLKNKLQMEVKVCRIGHNVSEI GTFINDNIIVNHHIKNQALTPVRRRLRKKGYIVGGVDNPRWQTGDALRGEIHKASYYGAITQFAKDDEGKVLMKEGRPQVNPT IKFVIRRELKYKKSAADSGFASWDDLGKAIVDKELFALMKGQFPAETSFKDACEQGIYMIKKGKNGMPDIKLHHIRHVRCEAP QSGLKIKEQTYKSEKEYKRYFYAAVGDLYAMCCYTNGKIREFRIYSLYDVSCHRKSDIEDIPEFITDKKGNRLMLDYKLRTGD MILLYKDNPAELYDLDNVNLSRRLYKINRFESQSNLVLMTHHLSTSKERGRSLGKTVDYQNLPESIRSSVKSLNFLIMGENRD FVIKNGKIIFNHR SEQ ID NO: 42 MLVSPISVDLGGKNTGFFSFTDSLDNSQSGTVIYDESFVLSQVGRRSKRHSKRNNLRNKLVKRLFLLILQEHHGLSIDVLPDE IRGLFNKRGYTYAGFELDEKKKDALESDTLKEFLSEKLQSIDRDSDVEDFLNQIASNAESFKDYKKGFEAVFASATHSPNKKL ELKDELKSEYGENAKELLAGLRVTKEILDEFDKQENQGNLPRAKYFEELGEYIATNEKVKSFFDSNSLKLTDMTKLIGNISNY QLKELRRYFNDKEMEKGDIWIPNKLHKITERFVRSWHPKNDADRQRRAELMKDLKSKEIMELLTTTEPVMTIPPYDDMNNRGA VKCQTLRLNEEYLDKHLPNWRDIAKRLNHGKFNDDLADSTVKGYSEDSTLLHRLLDTSKEIDIYELRGKKPNELLVKTLGQSD ANRLYGFAQNYYELIRQKVRAGIWVPVKNKDDSLNLEDNSNMLKRCNHNPPHKKNQIHNLVAGILGVKLDEAKFAEFEKELWS AKVGNKKLSAYCKNIEELRKTHGNTFKIDIEELRKKDPAELSKEEKAKLRLTDDVILNEWSQKIANFFDIDDKHRQRFNNLFS MAQLHTVIDTPRSGFSSTCKRCTAENRFRSETAFYNDETGEFHKKATATCQRLPADTQRPFSGKIERYIDKLGYELAKIKAKE LEGMEAKEIKVPIILEQNAFEYEESLRKSKTGSNDRVINSKKDRDGKKLAKAKENAEDRLKDKDKRIKAFSSGICPYCGDTIG DDGEIDHILPRSHTLKIYGTVFNPEGNLIYVHQKCNQAKADSIYKLSDIKAGVSAQWIEEQVANIKGYKTFSVLSAEQQKAFR YALFLQNDNEAYKKVVDWLRTDQSARVNGTQKYLAKKIQEKLTKMLPNKHLSFEFILADATEVSELRRQYARQNPLLAKAEKQ APSSHAIDAVMAFVARYQKVFKDGTPPNADEVAKLAMLDSWNPASNEPLTKGLSTNQKIEKMIKSGDYGQKNMREVFGKSIFG ENAIGERYKPIVVQEGGYYIGYPATVKKGYELKNCKVVTSKNDIAKLEKIIKNQDLISLKENQYIKIFSINKQTISELSNRYF NMNYKNLVERDKEIVGLLEFIVENCRYYTKKVDVKFAPKYIHETKYPFYDDWRRFDEAWRYLQENQNKTSSKDRFVIDKSSLN EYYQPDKNEYKLDVDTQPIWDDFCRWYFLDRYKTANDKKSIRIKARKTFSLLAESGVQGKVFRAKRKIPTGYAYQALPMDNNV IAGDYANILLEANSKTLSLVPKSGISIEKQLDKKLDVIKKTDVRGLAIDNNSFFNADFDTHGIRLIVENTSVKVGNFPISAID KSAKRMIFRALFEKEKGKRKKKTTISFKESGPVQDYLKVFLKKIVKIQLRTDGSISNIVVRKNAADFTLSFRSEHIQKLLK SEQ ID NO: 43 MAYRLGLDIGITSVGWAVVALEKDESGLKPVRIQDLGVRIFDKAEDSKTGASLALPRREARSARRRTRRRRHRLWRVKRLLEQ HGILSMEQIEALYAQRTSSPDVYALRVAGLDRCLIAEEIARVLIHIAHRRGFQSNRKSEIKDSDAGKLLKAVQENENLMQSKG YRTVAEMLVSEATKTDAEGKLVHGKKHGYVSNVRNKAGEYRHTVSRQAIVDEVRKIFAAQRALGNDVMSEELEDSYLKILCSQ RNFDDGPGGDSPYGHGSVSPDGVRQSIYERMVGSCTFETGEKRAPRSSYSFERFQLLTKVVNLRIYRQQEDGGRYPCELTQTE RARVIDCAYEQTKITYGKLRKLLDMKDTESFAGLTYGLNRSRNKTEDTVFVEMKFYHEVRKALQRAGVFIQDLSIETLDQIGW ILSVWKSDDNRRKKLSTLGLSDNVIEELLPLNGSKFGHLSLKAIRKILPFLEDGYSYDVACELAGYQFQGKTEYVKQRLLPPL GEGEVTNPVVRRALSQAIKVVNAVIRKHGSPESIHIELARELSKNLDERRKIEKAQKENQKNNEQIKDEIREILGSAHVTGRD IVKYKLFKQQQEFCMYSGEKLDVTRLFEPGYAEVDHIIPYGISFDDSYDNKVLVKTEQNRQKGNRTPLEYLRDKPEQKAKFIA LVESIPLSQKKKNHLLMDKRAIDLEQEGFRERNLSDTRYITRALMNHIQAWLLFDETASTRSKRVVCVNGAVTAYMRARWGLT KDRDAGDKHHAADAVVVACIGDSLIQRVTKYDKFKRNALADRNRYVQQVSKSEGITQYVDKETGEVFTWESFDERKFLPNEPL EPWPFFRDELLARLSDDPSKNIRAIGLLTYSETEQIDPIFVSRMPTRKVTGAAHKETIRSPRIVKVDDNKGTEIQVVVSKVAL TELKLTKDGEIKDYFRPEDDPRLYNTLRERLVQFGGDAKAAFKEPVYKISKDGSVRTPVRKVKIQEKLTLGVPVHGGRGIAEN GGMVRIDVFAKGGKYYFVPIYVADVLKRELPNRLATAHKPYSEWRVVDDSYQFKFSLYPNDAVMIKPSREVDITYKDRKEPVG CRIMYFVSANIASASISLRTHDNSGELEGLGIQGLEVFEKYVVGPLGDTHPVYKERRMPFRVERKMN SEQ ID NO: 44 MPVLSPLSPNAAQGRRRWSLALDIGEGSIGWAVAEVDAEGRVLQLTGTGVTLFPSAWSNENGTYVAHGAADRAVRGQQQRHDS RRRRLAGLARLCAPVLERSPEDLKDLTRTPPKADPRAIFFLRADAARRPLDGPELFRVLHHMAAHRGIRLAELQEVDPPPESD ADDAAPAATEDEDGTRRAAADERAFRRLMAEHMHRHGTQPTCGEIMAGRLRETPAGAQPVTRARDGLRVGGGVAVPTRALIEQ EFDAIRAIQAPRHPDLPWDSLRRLVLDQAPIAVPPATPCLFLEELRRRGETFQGRTITREAIDRGLTVDPLIQALRIRETVGN LRLHERITEPDGRQRYVPRAMPELGLSHGELTAPERDTLVRALMHDPDGLAAKDGRIPYTRLRKLIGYDNSPVCFAQERDTSG GGITVNPTDPLMARWIDGWVDLPLKARSLYVRDVVARGADSAALARLLAEGAHGVPPVAAAAVPAATAAILESDIMQPGRYSV CPWAAEAILDAWANAPTEGFYDVTRGLFGFAPGEIVLEDLRRARGALLAHLPRTMAAARTPNRAAQQRGPLPAYESVIPSQLI TSLRRAHKGRAADWSAADPEERNPFLRTWTGNAATDHILNQVRKTANEVITKYGNRRGWDPLPSRITVELAREAKHGVIRRNE IAKENRENEGRRKKESAALDTFCQDNTVSWQAGGLPKERAALRLRLAQRQEFFCPYCAERPKLRATDLFSPAETEIDHVIERR MGGDGPDNLVLAHKDCNNAKGKKTPHEHAGDLLDSPALAALWQGWRKENADRLKGKGHKARTPREDKDFMDRVGWRFEEDARA KAEENQERRGRRMLHDTARATRLARLYLAAAVMPEDPAEIGAPPVETPPSPEDPTGYTAIYRTISRVQPVNGSVTHMLRQRLL QRDKNRDYQTHHAEDACLLLLAGPAVVQAFNTEAAQHGADAPDDRPVDLMPTSDAYHQQRRARALGRVPLATVDAALADIVMP ESDRQDPETGRVHWRLTRAGRGLKRRIDDLTRNCVILSRPRRPSETGTPGALHNATHYGRREITVDGRTDTVVTQRMNARDLV ALLDNAKIVPAARLDAAAPGDTILKEICTEIADRHDRVVDPEGTHARRWISARLAALVPAHAEAVARDIAELADLDALADADR TPEQEARRSALRQSPYLGRAISAKKADGRARAREQEILTRALLDPHWGPRGLRHLIMREARAPSLVRIRANKTDAFGRPVPDA AVWVKTDGNAVSQLWRLTSVVTDDGRRIPLPKPIEKRIEISNLEYARLNGLDEGAGVTGNNAPPRPLRQDIDRLTPLWRDHGT APGGYLGTAVGELEDKARSALRGKAMRQTLTDAGITAEAGWRLDSEGAVCDLEVAKGDTVKKDGKTYKVGVITQGIFGMPVDA AGSAPRTPEDCEKFEEQYGIKPWKAKGIPLA SEQ ID NO: 45 MNYTEKEKLFMKYILALDIGIASVGWAILDKESETVIEAGSNIFPEASAADNQLRRDMRGAKRNNRRLKTRINDFIKLWENNN LSIPQFKSTEIVGLKVRAITEEITLDELYLILYSYLKHRGISYLEDALDDTVSGSSAYANGLKLNAKELETHYPCEIQQERLN TIGKYRGQSQIINENGEVLDLSNVFTIGAYRKEIQRVFEIQKKYHPELTDEFCDGYMLIFNRKRKYYEGPGNEKSRTDYGRFT TKLDANGNYITEDNIFEKLIGKCSVYPDELRAAAASYTAQEYNVLNDLNNLTINGRKLEENEKHEIVERIKSSNTINMRKIIS DCMGENIDDFAGARIDKSGKEIFHKFEVYNKMRKALLEIGIDISNYSREELDEIGYIMTINTDKEAMMEAFQKSWIDLSDDVK QCLINMRKTNGALFNKWQSFSLKIMNELIPEMYAQPKEQMTLLTEMGVTKGTQEEFAGLKYIPVDVVSEDIFNPVVRRSVRIS FKILNAVLKKYKALDTIVIEMPRDRNSEEQKKRINDSQKLNEKEMEYIEKKLAVTYGIKLSPSDFSSQKQLSLKLKLWNEQDG ICLYSGKTIDPNDIINNPQLFEIDHIIPRSISFDDARSNKVLVYRSENQKKGNQTPYYYLTHSHSEWSFEQYKATVMNLSKKK EYAISRKKIQNLLYSEDITKMDVLKGFINRNINDTSYASRLVLNTIQNFFMANEADTKVKVIKGSYTHQMRCNLKLDKNRDES YSHHAVDAMLIGYSELGYEAYHKLQGEFIDFETGEILRKDMWDENMSDEVYADYLYGKKWANIRNEVVKAEKNVKYWHYVMRK SNRGLCNQTIRGTREYDGKQYKINKLDIRTKEGIKVFAKLAFSKKDSDRERLLVYLNDRRTFDDLCKIYEDYSDAANPFVQYE KETGDIIRKYSKKHNGPRIDKLKYKDGEVGACIDISHKYGFEKGSKKVILESLVPYRMDVYYKEENHSYYLVGVKQSDIKFEK GRNVIDEEAYARILVNEKMIQPGQSRADLENLGFKFKLSFYKNDIIEYEKDGKIYTERLVSRTMPKQRNYIETKPIDKAKFEK QNLVGLGKTKFIKKYRYDILGNKYSCSEEKFTSFC SEQ ID NO: 46 MLRLYCANNLVLNNVQNLWKYLLLLIFDKKIIFLFKIKVILIRRYMENNNKEKIVIGFDLGVASVGWSIVNAETKEVIDLGVR LFSEPEKADYRRAKRTTRRLLRRKKFKREKFHKLILKNAEIFGLQSRNEILNVYKDQSSKYRNILKLKINALKEEIKPSELVW ILRDYLQNRGYFYKNEKLTDEFVSNSFPSKKLHEHYEKYGFFRGSVKLDNKLDNKKDKAKEKDEEEESDAKKESEELIFSNKQ WINEIVKVFENQSYLTESFKEEYLKLFNYVRPFNKGPGSKNSRTAYGVFSTDIDPETNKFKDYSNIWDKTIGKCSLFEEEIRA PKNLPSALIFNLQNEICTIKNEFTEFKNWWLNAEQKSEILKFVFTELFNWKDKKYSDKKFNKNLQDKIKKYLLNFALENFNLN EEILKNRDLENDTVLGLKGVKYYEKSNATADAALEFSSLKPLYVFIKFLKEKKLDLNYLLGLENTEILYFLDSIYLAISYSSD LKERNEWFKKLLKELYPKIKNNNLEIIENVEDIFEITDQEKFESFSKTHSLSREAFNHIIPLLLSNNEGKNYESLKHSNEELK KRTEKAELKAQQNQKYLKDNFLKEALVPLSVKTSVLQAIKIFNQIIKNFGKKYEISQVVIEMARELTKPNLEKLLNNATNSNI KILKEKLDQTEKFDDFTKKKFIDKIENSVVFRNKLFLWFEQDRKDPYTQLDIKINEIEDETEIDHVIPYSKSADDSWFNKLLV KKSTNQLKKNKTVWEYYQNESDPEAKWNKFVAWAKRIYLVQKSDKESKDNSEKNSIFKNKKPNLKFKNITKKLFDPYKDLGFL ARNLNDTRYATKVFRDQLNNYSKHHSKDDENKLFKWCMNGSITSFLRKSMVVRKNEEQVYRFNFWKKDRDQFFHHAVDASIIA IFSLLTKTLYNKLRVYESYDVQRREDGVYLINKETGEVKKADKDYWKDQHNFLKIRENAIEIKNVLNNVDFQNQVRYSRKANT KLNTQLFNETLYGVKEFENNFYKLEKVNLFSRKDLRKFILEDLNEESEKNKKNENGSRKRILTEKYIVDEILQILENEEFKDS KSDINALNKYMDSLPSKFSEFFSQDFINKCKKENSLILTFDAIKHNDPKKVIKIKNLKFFREDATLKNKQAVHKDSKNQIKSF YESYKCVGFIWLKNKNDLEESIFVPINSRVIHFGDKDKDIFDFDSYNKEKLLNEINLKRPENKKFNSINEIEFVKFVKPGALL LNFENQQIYYISTLESSSLRAKIKLLNKMDKGKAVSMKKITNPDEYKIIEHVNPLGINLNWTKKLENNN SEQ ID NO: 47 MLMSKHVLGLDLGVGSIGWCLIALDAQGDPAEILGMGSRVVPLNNATKAIEAFNAGAAFTASQERTARRTMRRGFARYQLRRY RLRRELEKVGMLPDAALIQLPLLELWELRERAATAGRRLTLPELGRVLCHINQKRGYRHVKSDAAAIVGDEGEKKKDSNSAYL AGIRANDEKLQAEHKTVGQYFAEQLRQNQSESPTGGISYRIKDQIFSRQCYIDEYDQIMAVQRVHYPDILTDEFIRMLRDEVI FMQRPLKSCKHLVSLCEFEKQERVMRVQQDDGKGGWQLVERRVKFGPKVAPKSSPLFQLCCIYEAVNNIRLTRPNGSPCDITP EERAKIVAHLQSSASLSFAALKKLLKEKALIADQLTSKSGLKGNSTRVALASALQPYPQYHHLLDMELETRMMTVQLTDEETG EVTEREVAVVTDSYVRKPLYRLWHILYSIEEREAMRRALITQLGMKEEDLDGGLLDQLYRLDFVKPGYGNKSAKFICKLLPQL QQGLGYSEACAAVGYRHSNSPTSEEITERTLLEKIPLLQRNELRQPLVEKILNQMINLVNALKAEYGIDEVRVELARELKMSR EERERMARNNKDREERNKGVAAKIRECGLYPTKPRIQKYMLWKEAGRQCLYCGRSIEEEQCLREGGMEVEHIIPKSVLYDDSY GNKTCACRRCNKEKGNRTALEYIRAKGREAEYMKRINDLLKEKKISYSKHQRLRWLKEDIPSDFLERQLRLTQYISRQAMAIL QQGIRRVSASEGGVTARLRSLWGYGKILHTLNLDRYDSMGETERVSREGEATEELHITNWSKRMDHRHHAIDALVVACTRQSY IQRLNRLSSEFGREDKKKEDQEAQEQQATETGRLSNLERWLTQRPHFSVRTVSDKVAEILISYRPGQRVVTRGRNIYRKKMAD GREVSCVQRGVLVPRGELMEASFYGKILSQGRVRIVKRYPLHDLKGEVVDPHLRELITTYNQELKSREKGAPIPPLCLDKDKK QEVRSVRCYAKTLSLDKAIPMCFDEKGEPTAFVKSASNHHLALYRTPKGKLVESIVTFWDAVDRARYGIPLVITHPREVMEQV LQRGDIPEQVLSLLPPSDWVFVDSLQQDEMVVIGLSDEELQRALEAQNYRKISEHLYRVQKMSSSYYVFRYHLETSVADDKNT SGRIPKFHRVQSLKAYEERNIRKVRVDLLGRISLL SEQ ID NO: 48 MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLVRRTNRQGRRLARRKKHRRVRLNRLFEESGLITDFTKIS INLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISYLDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERYQTYGQLR GDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQQEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGRYRTSGETLD NIFGILIGKCTFYPDEFRAAKASYTAQEFNLLNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLFKYIAKLLSCDVA DIKGYRIDKSGKAEIHTFEAYRKMKTLETLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFADGSFSQKQVDELVQFRKA NSSIFGKGWHNFSVKLMMELIPELYETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLTEEIYNPVVAKSVRQAIKIVNAAIK EYGDFDNIVIEMARETNEDDEKKAIQKIQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGERCLYTGK TISIHDLINNSNQFEVDHILPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDAWSFRELKAFVRESKTLSNKKKEYL LTEEDISKFDVRKKFIERNLVDTRYASRVVLNALQEHFRAHKIDTKVSVVRGQFTSQLRRHWGIEKTRDTYHHHAVDALIIAA SSQLNLWKKQKNTLVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLKSKEFEDSILFSYQVDSKFNRKISDATIYAT RQAKVGKDKADETYVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQTFEKVIEPILENYPNKQINEKGKEVPCNPFLKY KEEHGYIRKYSKKGNGPEIKSLKYYDSKLGNHIDITPKDSNNKVVLQSVSPWRADVYFNKTTGKYEILGLKYADLQFEKGTGT YKISQEKYNDIKKKEGVDSDSEFKFTLYKNDLLLVKDTETKEQQLFRFLSRTMPKQKHYVELKPYDKQKFEGGEALIKVLGNV ANSGQCKKGLGKSNISIYKVRTDVLGNQHIIKNEGDKPKLDF SEQ ID NO: 49 MNAEHGKEGLLIMEENFQYRIGLDIGITSVGWAVLQNNSQDEPVRITDLGVRIFDVAENPKNGDALAAPRRDARTTRRRLRRR RHRLERIKFLLQENGLIEMDSFMERYYKGNLPDVYQLRYEGLDRKLKDEELAQVLIHIAKHRGFRSTRKAETKEKEGGAVLKA TTENQKIMQEKGYRTVGEMLYLDEAFHTECLWNEKGYVLTPRNRPDDYKHTILRSMLVEEVHAIFAAQRAHGNQKATEGLEEA YVEIMTSQRSFDMGPGLQPDGKPSPYAMEGFGDRVGKCTFEKDEYRAPKATYTAELFVALQKINHTKLIDEFGTGRFFSEEER KTIIGLLLSSKELKYGTIRKKLNIDPSLKFNSLNYSAKKEGETEEERVLDTEKAKFASMFWTYEYSKCLKDRTEEMPVGEKAD LFDRIGEILTAYKNDDSRSSRLKELGLSGEEIDGLLDLSPAKYQRVSLKAMRKMQPYLEDGLIYDKACEAAGYDFRALNDGNK KHLLKGEEINAIVNDITNPVVKRSVSQTIKVINAIIQKYGSPQAVNIELAREMSKNFQDRTNLEKEMKKRQQENERAKQQIIE LGKQNPTGQDILKYRLWNDQGGYCLYSGKKIPLEELFDGGYDIDHILPYSITFDDSYRNKVLVTAQENRQKGNRTPYEYFGAD EKRWEDYEASVRLLVRDYKKQQKLLKKNFTEEERKEFKERNLNDTKYITRVVYNMIRQNLELEPFNHPEKKKQVWAVNGAVTS YLRKRWGLMQKDRSTDRHHAMDAVVIACCTDGMIHKISRYMQGRELAYSRNFKFPDEETGEILNRDNFTREQWDEKFGVKVPL PWNSFRDELDIRLLNEDPKNFLLTHADVQRELDYPGWMYGEEESPIEEGRYINYIRPLFVSRMPNHKVTGSAHDATIRSARDY ETRGVVITKVPLTDLKLNKDNEIEGYYDKDSDRLLYQALVRQLLLHGNDGKKAFAEDFHKPKADGTEGPVVRKVKIEKKQTSG VMVRGGTGIAANGEMVRIDVFRENGKYYFVPVYTADVVRKVLPNRAATHTKPYSEWRVMDDANFVFSLYSRDLIHVKSKKDIK TNLVNGGLLLQKEIFAYYTGADIATASIAGFANDSNFKFRGLGIQSLEIFEKCQVDILGNISVVRHENRQEFH SEQ ID NO: 50 MRVLGLDAGIASLGWALIEIEESNRGELSQGTIIGAGTWMFDAPEEKTQAGAKLKSEQRRTFRGQRRVVRRRRQRMNEVRRIL HSHGLLPSSDRDALKQPGLDPWRIRAEALDRLLGPVELAVALGHIARHRGFKSNSKGAKTNDPADDTSKMKRAVNETREKLAR FGSAAKMLVEDESFVLRQTPTKNGASEIVRRFRNREGDYSRSLLRDDLAAEMRALFTAQARFQSAIATADLQTAFTKAAFFQR PLQDSEKLVGPCPFEVDEKRAPKRGYSFELFRFLSRLNHVTLRDGKQERTLTRDELALAAADFGAAAKVSFTALRKKLKLPET TVFVGVKADEESKLDVVARSGKAAEGTARLRSVIVDALGELAWGALLCSPEKLDKIAEVISFRSDIGRISEGLAQAGCNAPLV DALTAAASDGRFDPFTGAGHISSKAARNILSGLRQGMTYDKACCAADYDHTASRERGAFDVGGHGREALKRILQEERISRELV GSPTARKALIESIKQVKAIVERYGVPDRIHVELARDVGKSIEEREEITRGIEKRNRQKDKLRGLFEKEVGRPPQDGARGKEEL LRFELWSEQMGRCLYTDDYISPSQLVATDDAVQVDHILPWSRFADDSYANKTLCMAKANQDKKGRTPYEWFKAEKTDTEWDAF IVRVEALADMKGFKKRNYKLRNAEEAAAKFRNRNLNDTRWACRLLAEALKQLYPKGEKDKDGKERRRVFSRPGALTDRLRRAW GLQWMKKSTKGDRIPDDRHHALDAIVIAATTESLLQRATREVQEIEDKGLHYDLVKNVTPPWPGFREQAVEAVEKVFVARAER RRARGKAHDATIRHIAVREGEQRVYERRKVAELKLADLDRVKDAERNARLIEKLRNWIEAGSPKDDPPLSPKGDPIFKVRLVT KSKVNIALDTGNPKRPGTVDRGEMARVDVFRKASKKGKYEYYLVPIYPHDIATMKTPPIRAVQAYKPEDEWPEMDSSYEFCWS LVPMTYLQVISSKGEIFEGYYRGMNRSVGAIQLSAHSNSSDVVQGIGARTLTEFKKFNVDRFGRKHEVERELRTWRGETWRGK AYI SEQ ID NO: 51 MGNYYLGLDVGIGSIGWAVINIEKKRIEDFNVRIFKSGEIQEKNRNSRASQQCRRSRGLRRLYRRKSHRKLRLKNYLSIIGLT TSEKIDYYYETADNNVIQLRNKGLSEKLTPEEIAACLIHICNNRGYKDFYEVNVEDIEDPDERNEYKEEHDSIVLISNLMNEG GYCTPAEMICNCREFDEPNSVYRKFHNSAASKNHYLITRHMLVKEVDLILENQSKYYGILDDKTIAKIKDIIFAQRDFEIGPG KNERFRRFTGYLDSIGKCQFFKDQERGSRFTVIADIYAFVNVLSQYTYTNNRGESVFDTSFANDLINSALKNGSMDKRELKAI AKSYHIDISDKNSDTSLTKCFKYIKVVKPLFEKYGYDWDKLIENYTDTDNNVLNRIGIVLSQAQTPKRRREKLKALNIGLDDG LINELTKLKLSGTANVSYKYMQGSIEAFCEGDLYGKYQAKFNKEIPDIDENAKPQKLPPFKNEDDCEFFKNPVVFRSINETRK LINAIIDKYGYPAAVNIETADELNKTFEDRAIDTKRNNDNQKENDRIVKEIIECIKCDEVHARHLIEKYKLWEAQEGKCLYSG ETITKEDMLRDKDKLFEVDHIVPYSLILDNTINNKALVYAEENQKKGQRTPLMYMNEAQAADYRVRVNTMFKSKKCSKKKYQY LMLPDLNDQELLGGWRSRNLNDTRYICKYLVNYLRKNLRFDRSYESSDEDDLKIRDHYRVFPVKSRFTSMFRRWWLNEKTWGR YDKAELKKLTYLDHAADAIIIANCRPEYVVLAGEKLKLNKMYHQAGKRITPEYEQSKKACIDNLYKLFRMDRRTAEKLLSGHG RLTPIIPNLSEEVDKRLWDKNIYEQFWKDDKDKKSCEELYRENVASLYKGDPKFASSLSMPVISLKPDHKYRGTITGEEAIRV KEIDGKLIKLKRKSISEITAESINSIYTDDKILIDSLKTIFEQADYKDVGDYLKKTNQHFFTTSSGKRVNKVTVIEKVPSRWL RKEIDDNNFSLLNDSSYYCIELYKDSKGDNNLQGIAMSDIVHDRKTKKLYLKPDFNYPDDYYTHVMYIFPGDYLRIKSTSKKS GEQLKFEGYFISVKNVNENSFRFISDNKPCAKDKRVSITKKDIVIKLAVDLMGKVQGENNGKGISCGEPLSLLKEKN SEQ ID NO: 52 MLSRQLLGASHLARPVSYSYNVQDNDVHCSYGERCFMRGKRYRIGIDVGLNSVGLAAVEVSDENSPVRLLNAQSVIHDGGVDP QKNKEAITRKNMSGVARRTRRMRRRKRERLHKLDMLLGKFGYPVIEPESLDKPFEEWHVRAELATRYIEDDELRRESISIALR HMARHRGWRNPYRQVDSLISDNPYSKQYGELKEKAKAYNDDATAAEEESTPAQLVVAMLDAGYAEAPRLRWRTGSKKPDAEGY LPVRLMQEDNANELKQIFRVQRVPADEWKPLFRSVFYAVSPKGSAEQRVGQDPLAPEQARALKASLAFQEYRIANVITNLRIK DASAELRKLTVDEKQSIYDQLVSPSSEDITWSDLCDFLGFKRSQLKGVGSLTEDGEERISSRPPRLTSVQRIYESDNKIRKPL VAWWKSASDNEHEAMIRLLSNTVDIDKVREDVAYASAIEFIDGLDDDALTKLDSVDLPSGRAAYSVETLQKLTRQMLTTDDDL HEARKTLFNVTDSWRPPADPIGEPLGNPSVDRVLKNVNRYLMNCQQRWGNPVSVNIEHVRSSFSSVAFARKDKREYEKNNEKR SIFRSSLSEQLRADEQMEKVRESDLRRLEAIQRQNGQCLYCGRTITFRTCEMDHIVPRKGVGSTNTRTNFAAVCAECNRMKSN TPFAIWARSEDAQTRGVSLAEAKKRVTMFTFNPKSYAPREVKAFKQAVIARLQQTEDDAAIDNRSIESVAWMADELHRRIDWY FNAKQYVNSASIDDAEAETMKTTVSVFQGRVTASARRAAGIEGKIHFIGQQSKTRLDRRHHAVDASVIAMMNTAAAQTLMERE SLRESQRLIGLMPGERSWKEYPYEGTSRYESFHLWLDNMDVLLELLNDALDNDRIAVMQSQRYVLGNSIAHDATIHPLEKVPL GSAMSADLIRRASTPALWCALTRLPDYDEKEGLPEDSHREIRVHDTRYSADDEMGFFASQAAQIAVQEGSADIGSAIHHARVY RCWKTNAKGVRKYFYGMIRVFQTDLLRACHDDLFTVPLPPQSISMRYGEPRVVQALQSGNAQYLGSLVVGDEIEMDFSSLDVD GQIGEYLQFFSQFSGGNLAWKHWVVDGFFNQTQLRIRPRYLAAEGLAKAFSDDVVPDGVQKIVTKQGWLPPVNTASKTAVRIV RRNAFGEPRLSSAHHMPCSWQWRHE SEQ ID NO: 53 MYSIGLDLGISSVGWSVIDERTGNVIDLGVRLFSAKNSEKNLERRTNRGGRRLIRRKTNRLKDAKKILAAVGFYEDKSLKNSC PYQLRVKGLTEPLSRGEIYKVTLHILKKRGISYLDEVDTEAAKESQDYKEQVRKNAQLLTKYTPGQIQLQRLKENNRVKTGIN AQGNYQLNVFKVSAYANELATILKTQQAFYPNELTDDWIALFVQPGIAEEAGLIYRKRPYYHGPGNEANNSPYGRWSDFQKTG EPATNIFDKLIGKDFQGELRASGLSLSAQQYNLLNDLTNLKIDGEVPLSSEQKEYILTELMTKEFTRFGVNDVVKLLGVKKER LSGWRLDKKGKPEIHTLKGYRNWRKIFAEAGIDLATLPTETIDCLAKVLTLNTEREGIENTLAFELPELSESVKLLVLDRYKE LSQSISTQSWHRFSLKTLHLLIPELMNATSEQNTLLEQFQLKSDVRKRYSEYKKLPTKDVLAEIYNPTVNKTVSQAFKVIDAL LVKYGKEQIRYITIEMPRDDNEEDEKKRIKELHAKNSQRKNDSQSYFMQKSGWSQEKFQTTIQKNRRFLAKLLYYYEQDGICA YTGLPISPELLVSDSTEIDHIIPISISLDDSINNKVLVLSKANQVKGQQTPYDAWMDGSFKKINGKFSNWDDYQKWVESRHFS HKKENNLLETRNIFDSEQVEKFLARNLNDTRYASRLVLNTLQSFFTNQETKVRVVNGSFTHTLRKKWGADLDKTRETHHHHAV DATLCAVTSFVKVSRYHYAVKEETGEKVMREIDFETGEIVNEMSYWEFKKSKKYERKTYQVKWPNFREQLKPVNLHPRIKFSH QVDRKANRKLSDATIYSVREKTEVKTLKSGKQKITTDEYTIGKIKDIYTLDGWEAFKKKQDKLLMKDLDEKTYERLLSIAETT PDFQEVEEKNGKVKRVKRSPFAVYCEENDIPAIQKYAKKNNGPLIRSLKYYDGKLNKHINITKDSQGRPVEKTKNGRKVTLQS LKPYRYDIYQDLETKAYYTVQLYYSDLRFVEGKYGITEKEYMKKVAEQTKGQVVRFCFSLQKNDGLEIEWKDSQRYDVRFYNF QSANSINFKGLEQEMMPAENQFKQKPYNNGAINLNIAKYGKEGKKLRKFNTDILGKKHYLFYEKEPKNIIK SEQ ID NO: 54 MYFYKNKENKLNKKVVLGLDLGIASVGWCLTDISQKEDNKFPIILHGVRLFETVDDSDDKLLNETRRKKRGQRRRNRRLFTRK RDFIKYLIDNNIIELEFDKNPKILVRNFIEKYINPFSKNLELKYKSVTNLPIGFHNLRKAAINEKYKLDKSELIVLLYFYLSL RGAFFDNPEDTKSKEMNKNEIEIFDKNESIKNAEFPIDKIIEFYKISGKIRSTINLKFGHQDYLKEIKQVFEKQNIDFMNYEK FAMEEKSFFSRIRNYSEGPGNEKSFSKYGLYANENGNPELIINEKGQKIYTKIFKTLWESKIGKCSYDKKLYRAPKNSFSAKV FDITNKLTDWKHKNEYISERLKRKILLSRFLNKDSKSAVEKILKEENIKFENLSEIAYNKDDNKINLPIINAYHSLTTIFKKH LINFENYLISNENDLSKLMSFYKQQSEKLFVPNEKGSYEINQNNNVLHIFDAISNILNKFSTIQDRIRILEGYFEFSNLKKDV KSSEIYSEIAKLREFSGTSSLSFGAYYKFIPNLISEGSKNYSTISYEEKALQNQKNNFSHSNLFEKTWVEDLIASPTVKRSLR QTMNLLKEIFKYSEKNNLEIEKIVVEVTRSSNNKHERKKIEGINKYRKEKYEELKKVYDLPNENTTLLKKLWLLRQQQGYDAY SLRKIEANDVINKPWNYDIDHIVPRSISFDDSFSNLVIVNKLDNAKKSNDLSAKQFIEKIYGIEKLKEAKENWGNWYLRNANG KAFNDKGKFIKLYTIDNLDEFDNSDFINRNLSDTSYITNALVNHLTFSNSKYKYSVVSVNGKQTSNLRNQIAFVGIKNNKETE REWKRPEGFKSINSNDFLIREEGKNDVKDDVLIKDRSFNGHHAEDAYFITIISQYFRSFKRIERLNVNYRKETRELDDLEKNN IKFKEKASFDNFLLINALDELNEKLNQMRFSRMVITKKNTQLFNETLYSGKYDKGKNTIKKVEKLNLLDNRTDKIKKIEEFFD EDKLKENELTKLHIFNHDKNLYETLKIIWNEVKIEIKNKNLNEKNYFKYFVNKKLQEGKISFNEWVPILDNDFKIIRKIRYIK FSSEEKETDEIIFSQSNFLKIDQRQNFSFHNTLYWVQIWVYKNQKDQYCFISIDARNSKFEKDEIKINYEKLKTQKEKLQIIN EEPILKINKGDLFENEEKELFYIVGRDEKPQKLEIKYILGKKIKDQKQIQKPVKKYFPNWKKVNLTYMGEIFKK SEQ ID NO: 55 MDNKNYRIGIDVGLNSIGFCAVEVDQHDTPLGFLNLSVYRHDAGIDPNGKKTNTTRLAMSGVARRTRRLFRKRKRRLAALDRF IEAQGWTLPDHADYKDPYTPWLVRAELAQTPIRDENDLHEKLAIAVRHIARHRGWRSPWVPVRSLHVEQPPSDQYLALKERVE AKTLLQMPEGATPAEMVVALDLSVDVNLRPKNREKTDTRPENKKPGFLGGKLMQSDNANELRKIAKIQGLDDALLRELIELVF AADSPKGASGELVGYDVLPGQHGKRRAEKAHPAFQRYRIASIVSNLRIRHLGSGADERLDVETQKRVFEYLLNAKPTADITWS DVAEEIGVERNLLMGTATQTADGERASAKPPVDVTNVAFATCKIKPLKEWWLNADYEARCVMVSALSHAEKLTEGTAAEVEVA EFLQNLSDEDNEKLDSFSLPIGRAAYSVDSLERLTKRMIENGEDLFEARVNEFGVSEDWRPPAEPIGARVGNPAVDRVLKAVN RYLMAAEAEWGAPLSVNIEHVREGFISKRQAVEIDRENQKRYQRNQAVRSQIADHINATSGVRGSDVTRYLAIQRQNGECLYC GTAITFVNSEMDHIVPRAGLGSTNTRDNLVATCERCNKSKSNKPFAVWAAECGIPGVSVAEALKRVDFWIADGFASSKEHREL QKGVKDRLKRKVSDPEIDNRSMESVAWMARELAHRVQYYFDEKHTGTKVRVFRGSLTSAARKASGFESRVNFIGGNGKTRLDR RHHAMDAATVAMLRNSVAKTLVLRGNIRASERAIGAAETWKSFRGENVADRQIFESWSENMRVLVEKFNLALYNDEVSIFSSL RLQLGNGKAHDDTITKLQMHKVGDAWSLTEIDRASTPALWCALTRQPDFTWKDGLPANEDRTIIVNGTHYGPLDKVGIFGKAA ASLLVRGGSVDIGSAIHHARIYRIAGKKPTYGMVRVFAPDLLRYRNEDLFNVELPPQSVSMRYAEPKVREAINEGKAEYLGWL VVGDELLLDLSSETSGQIAELQQDFPGTTHWTVAGFFSPSRLRLRPVYLAQEGLGEDVSEGSKSIIAGQGWRPAVNKVFGSAM PEVIRRDGLGRKRRFSYSGLPVSWQG SEQ ID NO: 56 MRLGLDIGTSSIGWWLYETDGAGSDARITGVVDGGVRIFSDGRDPKSGASLAVDRRAARAMRRRRDRYLRRRATLMKVLAETG LMPADPAEAKALEALDPFALRAAGLDEPLPLPHLGRALFHLNQRRGFKSNRKTDRGDNESGKIKDATARLDMEMMANGARTYG EFLHKRRQKATDPRHVPSVRTRLSIANRGGPDGKEEAGYDFYPDRRHLEEEFHKLWAAQGAHHPELTETLRDLLFEKIFFQRP LKEPEVGLCLFSGHHGVPPKDPRLPKAHPLTQRRVLYETVNQLRVTADGREARPLTREERDQVIHALDNKKPTKSLSSMVLKL PALAKVLKLRDGERFTLETGVRDAIACDPLRASPAHPDRFGPRWSILDADAQWEVISRIRRVQSDAEHAALVDWLTEAHGLDR AHAEATAHAPLPDGYGRLGLTATTRILYQLTADVVTYADAVKACGWHHSDGRTGECFDRLPYYGEVLERHVIPGSYHPDDDDI TRFGRITNPTVHIGLNQLRRLVNRIIETHGKPHQIVVELARDLKKSEEQKRADIKRIRDTTEAAKKRSEKLEELEIEDNGRNR MLLRLWEDLNPDDAMRRFCPYTGTRISAAMIFDGSCDVDHILPYSRTLDDSFPNRTLCLREANRQKRNQTPWQAWGDTPHWHA IAANLKNLPENKRWRFAPDAMTRFEGENGFLDRALKDTQYLARISRSYLDTLFTKGGHVWVVPGRFTEMLRRHWGLNSLLSDA GRGAVKAKNRTDHRHHAIDAAVIAATDPGLLNRISRAAGQGEAAGQSAELIARDTPPPWEGFRDDLRVRLDRIIVSHRADHGR IDHAARKQGRDSTAGQLHQETAYSIVDDIHVASRTDLLSLKPAQLLDEPGRSGQVRDPQLRKALRVATGGKTGKDFENALRYF ASKPGPYQAIRRVRIIKPLQAQARVPVPAQDPIKAYQGGSNHLFEIWRLPDGEIEAQVITSFEAHTLEGEKRPHPAAKRLLRV HKGDMVALERDGRRVVGHVQKMDIANGLFIVPHNEANADTRNNDKSDPFKWIQIGARPAIASGIRRVSVDEIGRLRDGGTRPI SEQ ID NO: 57 MLHCIAVIRVPPSEEPGFFETHADSCALCHHGCMTYAANDKAIRYRVGIDVGLRSIGFCAVEVDDEDHPIRILNSVVHVHDAG TGGPGETESLRKRSGVAARARRRGRAEKQRLKKLDVLLEELGWGVSSNELLDSHAPWHIRKRLVSEYIEDETERRQCLSVAMA HIARHRGWRNSFSKVDTLLLEQAPSDRMQGLKERVEDRTGLQFSEEVTQGELVATLLEHDGDVTIRGFVRKGGKATKVHGVLE GKYMQSDLVAELRQICRTQRVSETTFEKLVLSIFHSKEPAPSAARQRERVGLDELQLALDPAAKQPRAERAHPAFQKFKVVAT LANMRIREQSAGERSLTSEELNRVARYLLNHTESESPTWDDVARKLEVPRHRLRGSSRASLETGGGLTYPPVDDTTVRVMSAE VDWLADWWDCANDESRGHMIDAISNGCGSEPDDVEDEEVNELISSATAEDMLKLELLAKKLPSGRVAYSLKTLREVTAAILET GDDLSQAITRLYGVDPGWVPTPAPIEAPVGNPSVDRVLKQVARWLKFASKRWGVPQTVNIEHTREGLKSASLLEEERERWERF EARREIRQKEMYKRLGISGPFRRSDQVRYEILDLQDCACLYCGNEINFQTFEVDHIIPRVDASSDSRRTNLAAVCHSCNSAKG GLAFGQWVKRGDCPSGVSLENAIKRVRSWSKDRLGLTEKAMGKRKSEVISRLKTEMPYEEFDGRSMESVAWMAIELKKRIEGY FNSDRPEGCAAVQVNAYSGRLTACARRAAHVDKRVRLIRLKGDDGHHKNRFDRRNHAMDALVIALMTPAIARTIAVREDRREA QQLTRAFESWKNFLGSEERMQDRWESWIGDVEYACDRLNELIDADKIPVTENLRLRNSGKLHADQPESLKKARRGSKRPRPQR YVLGDALPADVINRVTDPGLWTALVRAPGFDSQLGLPADLNRGLKLRGKRISADFPIDYFPTDSPALAVQGGYVGLEFHHARL YRIIGPKEKVKYALLRVCAIDLCGIDCDDLFEVELKPSSISMRTADAKLKEAMGNGSAKQIGWLVLGDEIQIDPTKFPKQSIG KFLKECGPVSSWRVSALDTPSKITLKPRLLSNEPLLKTSRVGGHESDLVVAECVEKIMKKTGWVVEINALCQSGLIRVIRRNA LGEVRTSPKSGLPISLNLR SEQ ID NO: 58 MRYRVGLDLGTASVGAAVFSMDEQGNPMELIWHYERLFSEPLVPDMGQLKPKKAARRLARQQRRQIDRRASRLRRIAIVSRRL GIAPGRNDSGVHGNDVPTLRAMAVNERIELGQLRAVLLRMGKKRGYGGTFKAVRKVGEAGEVASGASRLEEEMVALASVQNKD SVTVGEYLAARVEHGLPSKLKVAANNEYYAPEYALFRQYLGLPAIKGRPDCLPNMYALRHQIEHEFERIWATQSQFHDVMKDH GVKEEIRNAIFFQRPLKSPADKVGRCSLQTNLPRAPRAQIAAQNFRIEKQMADLRWGMGRRAEMLNDHQKAVIRELLNQQKEL SFRKIYKELERAGCPGPEGKGLNMDRAALGGRDDLSGNTTLAAWRKLGLEDRWQELDEVTQIQVINFLADLGSPEQLDTDDWS CRFMGKNGRPRNFSDEFVAFMNELRMTDGFDRLSKMGFEGGRSSYSIKALKALTEWMIAPHWRETPETHRVDEEAAIRECYPE SLATPAQGGRQSKLEPPPLTGNEVVDVALRQVRHTINMMIDDLGSVPAQIVVEMAREMKGGVTRRNDIEKQNKRFASERKKAA QSIEENGKTPTPARILRYQLWIEQGHQCPYCESNISLEQALSGAYTNFEHILPRTLTQIGRKRSELVLAHRECNDEKGNRTPY QAFGHDDRRWRIVEQRANALPKKSSRKTRLLLLKDFEGEALTDESIDEFADRQLHESSWLAKVTTQWLSSLGSDVYVSRGSLT AELRRRWGLDTVIPQVRFESGMPVVDEEGAEITPEEFEKFRLQWEGHRVTREMRTDRRPDKRIDHRHHLVDAIVTALTSRSLY QQYAKAWKVADEKQRHGRVDVKVELPMPILTIRDIALEAVRSVRISHKPDRYPDGRFFEATAYGIAQRLDERSGEKVDWLVSR KSLTDLAPEKKSIDVDKVRANISRIVGEAIRLHISNIFEKRVSKGMTPQQALREPIEFQGNILRKVRCFYSKADDCVRIEHSS RRGHHYKMLLNDGFAYMEVPCKEGILYGVPNLVRPSEAVGIKRAPESGDFIRFYKGDTVKNIKTGRVYTIKQILGDGGGKLIL TPVTETKPADLLSAKWGRLKVGGRNIHLLRLCAE SEQ ID NO: 59 MIGEHVRGGCLFDDHWTPNWGAFRLPNTVRTFTKAENPKDGSSLAEPRRQARGLRRRLRRKTQRLEDLRRLLAKEGVLSLSDL ETLFRETPAKDPYQLRAEGLDRPLSFPEWVRVLYHITKHRGFQSNRRNPVEDGQERSRQEEEGKLLSGVGENERLLREGGYRT AGEMLARDPKFQDHRRNRAGDYSHTLSRSLLLEEARRLFQSQRTLGNPHASSNLEEAFLHLVAFQNPFASGEDIRNKAGHCSL EPDQIRAPRRSASAETFMLLQKTGNLRLIHRRTGEERPLTDKEREQIHLLAWKQEKVIHKTLRRHLEIPEEWLFTGLPYHRSG DKAEEKLFVHLAGIHEIRKALDKGPDPAVWDTLRSRRDLLDSIADTLTFYKNEDEILPRLESLGLSPENARALAPLSFSGTAH LSLSALGKLLPHLEEGKSYTQARADAGYAAPPPDRHPKLPPLEEADWRNPVVFRALTQTRKVVNALVRRYGPPWCIHLETARE LSQPAKVRRRIETEQQANEKKKQQAEREFLDIVGTAPGPGDLLKMRLWREQGGFCPYCEEYLNPTRLAEPGYAEMDHILPYSR SLDNGWHNRVLVHGKDNRDKGNRTPFEAFGGDTARWDRLVAWVQASHLSAPKKRNLLREDFGEEAERELKDRNLTDTRFITKT AATLLRDRLTFHPEAPKDPVMTLNGRLTAFLRKQWGLHKNRKNGDLHHALDAAVLAVASRSFVYRLSSHNAAWGELPRGREAE NGFSLPYPAFRSEVLARLCPTREEILLRLDQGGVGYDEAFRNGLRPVFVSRAPSRRLRGKAHMETLRSPKWKDHPEGPRTASR IPLKDLNLEKLERMVGKDRDRKLYEALRERLAAFGGNGKKAFVAPFRKPCRSGEGPLVRSLRIFDSGYSGVELRDGGEVYAVA DHESMVRVDVYAKKNRFYLVPVYVADVARGIVKNRAIVAHKSEEEWDLVDGSFDFRFSLFPGDLVEIEKKDGAYLGYYKSCHR GDGRLLLDRHDRMPRESDCGTFYVSTRKDVLSMSKYQVDPLGEIRLVGSEKPPFVL SEQ ID NO: 60 MEKKRKVTLGFDLGIASVGWAIVDSETNQVYKLGSRLFDAPDTNLERRTQRGTRRLLRRRKYRNQKFYNLVKRTEVFGLSSRE AIENRFRELSIKYPNIIELKTKALSQEVCPDEIAWILHDYLKNRGYFYDEKETKEDFDQQTVESMPSYKLNEFYKKYGYFKGA LSQPTESEMKDNKDLKEAFFFDFSNKEWLKEINYFFNVQKNILSETFIEEFKKIFSFTRDISKGPGSDNMPSPYGIFGEFGDN GQGGRYEHIWDKNIGKCSIFTNEQRAPKYLPSALIFNFLNELANIRLYSTDKKNIQPLWKLSSVDKLNILLNLFNLPISEKKK KLTSTNINDIVKKESIKSIMISVEDIDMIKDEWAGKEPNVYGVGLSGLNIEESAKENKFKFQDLKILNVLINLLDNVGIKFEF KDRNDIIKNLELLDNLYLFLIYQKESNNKDSSIDLFIAKNESLNIENLKLKLKEFLLGAGNEFENHNSKTHSLSKKAIDEILP KLLDNNEGWNLEAIKNYDEEIKSQIEDNSSLMAKQDKKYLNDNFLKDAILPPNVKVTFQQAILIFNKIIQKFSKDFEIDKVVI ELAREMTQDQENDALKGIAKAQKSKKSLVEERLEANNIDKSVFNDKYEKLIYKIFLWISQDFKDPYTGAQISVNEIVNNKVEI DHIIPYSLCFDDSSANKVLVHKQSNQEKSNSLPYEYIKQGHSGWNWDEFTKYVKRVFVNNVDSILSKKERLKKSENLLTASYD GYDKLGFLARNLNDTRYATILFRDQLNNYAEHHLIDNKKMFKVIAMNGAVTSFIRKNMSYDNKLRLKDRSDFSHHAYDAAIIA LFSNKTKTLYNLIDPSLNGIISKRSEGYWVIEDRYTGEIKELKKEDWTSIKNNVQARKIAKEIEEYLIDLDDEVFFSRKTKRK TNRQLYNETIYGIATKTDEDGITNYYKKEKFSILDDKDIYLRLLREREKFVINQSNPEVIDQIIEIIESYGKENNIPSRDEAI NIKYTKNKINYNLYLKQYMRSLTKSLDQFSEEFINQMIANKTFVLYNPTKNTTRKIKFLRLVNDVKINDIRKNQVINKFNGKN NEPKAFYENINSLGAIVFKNSANNFKTLSINTQIAIFGDKNWDIEDFKTYNMEKIEKYKEIYGIDKTYNFHSFIFPGTILLDK QNKEFYYISSIQTVRDIIEIKFLNKIEFKDENKNQDTSKTPKRLMFGIKSIMNNYEQVDISPFGINKKIFE SEQ ID NO: 61 MGYRIGLDVGITSTGYAVLKTDKNGLPYKILTLDSVIYPRAENPQTGASLAEPRRIKRGLRRRTRRTKFRKQRTQQLFIHSGL LSKPEIEQILATPQAKYSVYELRVAGLDRRLTNSELFRVLYFFIGHRGFKSNRKAELNPENEADKKQMGQLLNSIEEIRKAIA EKGYRTVGELYLKDPKYNDHKRNKGYIDGYLSTPNRQMLVDEIKQILDKQRELGNEKLTDEFYATYLLGDENRAGIFQAQRDF DEGPGAGPYAGDQIKKMVGKDIFEPTEDRAAKATYTFQYFNLLQKMTSLNYQNTTGDTWHTLNGLDRQAIIDAVFAKAEKPTK TYKPTDFGELRKLLKLPDDARFNLVNYGSLQTQKEIETVEKKTRFVDFKAYHDLVKVLPEEMWQSRQLLDHIGTALTLYSSDK RRRRYFAEELNLPAELIEKLLPLNFSKFGHLSIKSMQNIIPYLEMGQVYSEATTNTGYDFRKKQISKDTIREEITNPVVRRAV TKTIKIVEQIIRRYGKPDGINIELARELGRNFKERGDIQKRQDKNRQTNDKIAAELTELGIPVNGQNIIRYKLHKEQNGVDPY TGDQIPFERAFSEGYEVDHIIPYSISWDDSYTNKVLTSAKCNREKGNRIPMVYLANNEQRLNALTNIADNIIRNSRKRQKLLK QKLSDEELKDWKQRNINDTRFITRVLYNYFRQAIEFNPELEKKQRVLPLNGEVTSKIRSRWGFLKVREDGDLHHAIDATVIAA ITPKFIQQVTKYSQHQEVKNNQALWHDAEIKDAEYAAEAQRMDADLFNKIFNGFPLPWPEFLDELLARISDNPVEMMKSRSWN TYTPIEIAKLKPVFVVRLANHKISGPAHLDTIRSAKLFDEKGIVLSRVSITKLKINKKGQVATGDGIYDPENSNNGDKVVYSA IRQALEAHNGSGELAFPDGYLEYVDHGTKKLVRKVRVAKKVSLPVRLKNKAAADNGSMVRIDVFNTGKKFVFVPIYIKDTVEQ VLPNKAIARGKSLWYQITESDQFCFSLYPGDMVHIESKTGIKPKYSNKENNTSVVPIKNFYGYFDGADIATASILVRAHDSSY TARSIGIAGLLKFEKYQVDYFGRYHKVHEKKRQLFVKRDE SEQ ID NO: 62 MQKNINTKQNHIYIKQAQKIKEKLGDKPYRIGLDLGVGSIGFAIVSMEENDGNVLLPKEIIMVGSRIFKASAGAADRKLSRGQ RNNHRHTRERMRYLWKVLAEQKLALPVPADLDRKENSSEGETSAKRFLGDVLQKDIYELRVKSLDERLSLQELGYVLYHIAGH RGSSAIRTFENDSEEAQKENTENKKIAGNIKRLMAKKNYRTYGEYLYKEFFENKEKHKREKISNAANNHKFSPTRDLVIKEAE AILKKQAGKDGFHKELTEEYIEKLTKAIGYESEKLIPESGFCPYLKDEKRLPASHKLNEERRLWETLNNARYSDPIVDIVTGE ITGYYEKQFTKEQKQKLFDYLLTGSELTPAQTKKLLGLKNTNFEDIILQGRDKKAQKIKGYKLIKLESMPFWARLSEAQQDSF LYDWNSCPDEKLLTEKLSNEYHLTEEEIDNAFNEIVLSSSYAPLGKSAMLIILEKIKNDLSYTEAVEEALKEGKLTKEKQAIK DRLPYYGAVLQESTQKIIAKGFSPQFKDKGYKTPHTNKYELEYGRIANPVVHQTLNELRKLVNEIIDILGKKPCEIGLETARE LKKSAEDRSKLSREQNDNESNRNRIYEIYIRPQQQVIITRRENPRNYILKFELLEEQKSQCPFCGGQISPNDIINNQADIEHL FPIAESEDNGRNNLVISHSACNADKAKRSPWAAFASAAKDSKYDYNRILSNVKENIPHKAWRFNQGAFEKFIENKPMAARFKT DNSYISKVAHKYLACLFEKPNIICVKGSLTAQLRMAWGLQGLMIPFAKQLITEKESESFNKDVNSNKKIRLDNRHHALDAIVI AYASRGYGNLLNKMAGKDYKINYSERNWLSKILLPPNNIVWENIDADLESFESSVKTALKNAFISVKHDHSDNGELVKGTMYK IFYSERGYTLTTYKKLSALKLTDPQKKKTPKDFLETALLKFKGRESEMKNEKIKSAIENNKRLFDVIQDNLEKAKKLLEEENE KSKAEGKKEKNINDASIYQKAISLSGDKYVQLSKKEPGKFFAISKPTPTTTGYGYDTGDSLCVDLYYDNKGKLCGEIIRKIDA QQKNPLKYKEQGFTLFERIYGGDILEVDFDIHSDKNSFRNNTGSAPENRVFIKVGTFTEITNNNIQIWFGNIIKSTGGQDDSF TINSMQQYNPRKLILSSCGFIKYRSPILKNKEG SEQ ID NO: 63 MAAFKPNPINYILGLDIGIASVGWAMVEIDEDENPICLIDLGVRVFERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRAR RLLKREGVLQAADFDENGLIKSLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVADNA HALQTGDFRTPAELALNKFEKESGHIRNQRGDYSHTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLMTQRPALSGD AVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFF KGLRYGKDNAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLKDRIQPEILEALLKHI SFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTEEKIYLPPIFADEIRNPVVLRALSQARKVINGVVRRYGSP ARIHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLGRLNEK GYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQRILLQKFDEDGFKE RNLNDTRYVNRFLCQFVADRMRLTGKGKKRVFASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRY KEMNAFDGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAEKLSSRPEAVHEYVTPLFV SRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKDLEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKA GNRTQQVKAVRVEQVQKTGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQLIDDSFN FKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILEGIGVKTALSFQKYQIDELGKEIRPCRLKKRP PVR SEQ ID NO: 64 MQTTNLSYILGLDLGIASVGWAVVEINENEDPIGLIDVGVRIFERAEVPKTGESLALSRRLARSTRRLIRRRAHRLLLAKRFL KREGILSTIDLEKGLPNQAWELRVAGLERRLSAIEWGAVLLHLIKHRGYLSKRKNESQTNNKELGALLSGVAQNHQLLQSDDY RTPAELALKKFAKEEGHIRNQRGAYTHTFNRLDLLAELNLLFAQQHQFGNPHCKEHIQQYMTELLMWQKPALSGEAILKMLGK CTHEKNEFKAAKHTYSAERFVWLTKLNNLRILEDGAERALNEEERQLLINHPYEKSKLTYAQVRKLLGLSEQAIFKHLRYSKE NAESATFMELKAWHAIRKALENQGLKDTWQDLAKKPDLLDEIGTAFSLYKTDEDIQQYLTNKVPNSVINALLVSLNFDKFIEL SLKSLRKILPLMEQGKRYDQACREIYGHHYGEANQKTSQLLPAIPAQEIRNPVVLRTLSQARKVINAIIRQYGSPARVHIETG RELGKSFKERREIQKQQEDNRTKRESAVQKFKELFSDFSSEPKSKDILKFRLYEQQHGKCLYSGKEINIHRLNEKGYVEIDHA LPFSRTWDDSFNNKVLVLASENQNKGNQTPYEWLQGKINSERWKNFVALVLGSQCSAAKKQRLLTQVIDDNKFIDRNLNDTRY IARFLSNYIQENLLLVGKNKKNVFTPNGQITALLRSRWGLIKARENNNRHHALDAIVVACATPSMQQKITRFIRFKEVHPYKI ENRYEMVDQESGEIISPHFPEPWAYFRQEVNIRVFDNHPDTVLKEMLPDRPQANHQFVQPLFVSRAPTRKMSGQGHMETIKSA KRLAEGISVLRIPLTQLKPNLLENMVNKEREPALYAGLKARLAEFNQDPAKAFATPFYKQGGQQVKAIRVEQVQKSGVLVREN NGVADNASIVRTDVFIKNNKFFLVPIYTWQVAKGILPNKAIVAHKNEDEWEEMDEGAKFKFSLFPNDLVELKTKKEYFFGYYI GLDRATGNISLKEHDGEISKGKDGVYRVGVKLALSFEKYQVDELGKNRQICRPQQRQPVR SEQ ID NO: 65 MGIRFAFDLGTNSIGWAVWRTGPGVFGEDTAASLDGSGVLIFKDGRNPKDGQSLATMRRVPRQSRKRRDRFVLRRRDLLAALR KAGLFPVDVEEGRRLAATDPYHLRAKALDESLTPHEMGRVIFHLNQRRGFRSNRKADRQDREKGKIAEGSKRLAETLAATNCR TLGEFLWSRHRGTPRTRSPTRIRMEGEGAKALYAFYPTREMVRAEFERLWTAQSRFAPDLLTPERHEEIAGILFRQRDLAPPK IGCCTFEPSERRLPRALPSVEARGIYERLAHLRITTGPVSDRGLTRPERDVLASALLAGKSLTFKAVRKTLKILPHALVNFEE AGEKGLDGALTAKLLSKPDHYGAAWHGLSFAEKDTFVGKLLDEADEERLIRRLVTENRLSEDAARRCASIPLADGYGRLGRTA NTEILAALVEETDETGTVVTYAEAVRRAGERTGRNWHHSDERDGVILDRLPYYGEILQRHVVPGSGEPEEKNEAARWGRLANP TVHIGLNQLRKVVNRLIAAHGRPDQIVVELARELKLNREQKERLDRENRKNREENERRTAILAEHGQRDTAENKIRLRLFEEQ ARANAGIALCPYTGRAIGIAELFTSEVEIDHILPVSLTLDDSLANRVLCRREANREKRRQTPFQAFGATPAWNDIVARAAKLP PNKRWRFDPAALERFEREGGFLGRQLNETKYLSRLAKIYLGKICDPDRVYVTPGTLTGLLRARWGLNSILSDSNFKNRSDHRH HAVDAVVIGVLTRGMIQRIAHDAARAEDQDLDRVFRDVPVPFEDFRDHVRERVSTITVAVKPEHGKGGALHEDTSYGLVPDTD PNAALGNLVVRKPIRSLTAGEVDRVRDRALRARLGALAAPFRDESGRVRDAKGLAQALEAFGAENGIRRVRILKPDASVVTIA DRRTGVPYRAVAPGENHHVDIVQMRDGSWRGFAASVFEVNRPGWRPEWEVKKLGGKLVMRLHKGDMVELSDKDGQRRVKVVQQ IEISANRVRLSPHNDGGKLQDRHADADDPFRWDLATIPLLKDRGCVAVRVDPIGVVTLRRSNV SEQ ID NO: 66 MMEVFMGRLVLGLDIGITSVGFGIIDLDESEIVDYGVRLFKEGTAAENETRRTKRGGRRLKRRRVTRREDMLHLLKQAGIIST SFHPLNNPYDVRVKGLNERLNGEELATALLHLCKHRGSSVETIEDDEAKAKEAGETKKVLSMNDQLLKSGKYVCEIQKERLRT NGHIRGHENNFKTRAYVDEAFQILSHQDLSNELKSAIITIISRKRMYYDGPGGPLSPTPYGRYTYFGQKEPIDLIEKMRGKCS LFPNEPRAPKLAYSAELFNLLNDLNNLSIEGEKLTSEQKAMILKIVHEKGKITPKQLAKEVGVSLEQIRGFRIDTKGSPLLSE LTGYKMIREVLEKSNDEHLEDHVFYDEIAEILTKTKDIEGRKKQISELSSDLNEESVHQLAGLTKFTAYHSLSFKALRLINEE MLKTELNQMQSITLFGLKQNNELSVKGMKNIQADDTAILSPVAKRAQRETFKVVNRLREIYGEFDSIVVEMAREKNSEEQRKA IRERQKFFEMRNKQVADIIGDDRKINAKLREKLVLYQEQDGKTAYSLEPIDLKLLIDDPNAYEVDHIIPISISLDDSITNKVL VTHRENQEKGNLTPISAFVKGRFTKGSLAQYKAYCLKLKEKNIKTNKGYRKKVEQYLLNENDIYKYDIQKEFINRNLVDTSYA SRVVLNTLTTYFKQNEIPTKVFTVKGSLTNAFRRKINLKKDRDEDYGHHAIDALIIASMPKMRLLSTIFSRYKIEDIYDESTG EVFSSGDDSMYYDDRYFAFIASLKAIKVRKFSHKIDTKPNRSVADETIYSTRVIDGKEKVVKKYKDIYDPKFTALAEDILNNA YQEKYLMALHDPQTFDQIVKVVNYYFEEMSKSEKYFTKDKKGRIKISGMNPLSLYRDEHGMLKKYSKKGDGPAITQMKYFDGV LGNHIDISAHYQVRDKKVVLQQISPYRTDFYYSKENGYKFVTIRYKDVRWSEKKKKYVIDQQDYAMKKAEKKIDDTYEFQFSM HRDELIGITKAEGEALIYPDETWHNFNFFFHAGETPEILKFTATNNDKSNKIEVKPIHCYCKMRLMPTISKKIVRIDKYATDV VGNLYKVKKNTLKFEFD SEQ ID NO: 67 MKKILGVDLGITSFGYAILQETGKDLYRCLDNSVVMRNNPYDEKSGESSQSIRSTQKSMRRLIEKRKKRIRCVAQTMERYGIL DYSETMKINDPKNNPIKNRWQLRAVDAWKRPLSPQELFAIFAHMAKHRGYKSIATEDLIYELELELGLNDPEKESEKKADERR QVYNALRHLEELRKKYGGETIAQTIHRAVEAGDLRSYRNHDDYEKMIRREDIEEEIEKVLLRQAELGALGLPEEQVSELIDEL KACITDQEMPTIDESLFGKCTFYKDELAAPAYSYLYDLYRLYKKLADLNIDGYEVTQEDREKVIEWVEKKIAQGKNLKKITHK DLRKILGLAPEQKIFGVEDERIVKGKKEPRTFVPFFFLADIAKFKELFASIQKHPDALQIFRELAEILQRSKTPQEALDRLRA LMAGKGIDTDDRELLELFKNKRSGTRELSHRYILEALPLFLEGYDEKEVQRILGFDDREDYSRYPKSLRHLHLREGNLFEKEE NPINNHAVKSLASWALGLIADLSWRYGPFDEIILETTRDALPEKIRKEIDKAMREREKALDKIIGKYKKEFPSIDKRLARKIQ LWERQKGLDLYSGKVINLSQLLDGSADIEHIVPQSLGGLSTDYNTIVTLKSVNAAKGNRLPGDWLAGNPDYRERIGMLSEKGL IDWKKRKNLLAQSLDEIYTENTHSKGIRATSYLEALVAQVLKRYYPFPDPELRKNGIGVRMIPGKVTSKTRSLLGIKSKSRET NFHHAEDALILSTLTRGWQNRLHRMLRDNYGKSEAELKELWKKYMPHIEGLTLADYIDEAFRRFMSKGEESLFYRDMFDTIRS ISYWVDKKPLSASSHKETVYSSRHEVPTLRKNILEAFDSLNVIKDRHKLTTEEFMKRYDKEIRQKLWLHRIGNTNDESYRAVE ERATQIAQILTRYQLMDAQNDKEIDEKFQQALKELITSPIEVTGKLLRKMRFVYDKLNAMQIDRGLVETDKNMLGIHISKGPN EKLIFRRMDVNNAHELQKERSGILCYLNEMLFIFNKKGLIHYGCLRSYLEKGQGSKYIALFNPRFPANPKAQPSKFTSDSKIK QVGIGSATGIIKAHLDLDGHVRSYEVFGTLPEGSIEWFKEESGYGRVEDDPHH SEQ ID NO: 68 MRPIEPWILGLDIGTDSLGWAVFSCEEKGPPTAKELLGGGVRLFDSGRDAKDHTSRQAERGAFRRARRQTRTWPWRRDRLIAL FQAAGLTPPAAETRQIALALRREAVSRPLAPDALWAALLHLAHHRGFRSNRIDKRERAAAKALAKAKPAKATAKATAPAKEAD DEAGFWEGAEAALRQRMAASGAPTVGALLADDLDRGQPVRMRYNQSDRDGVVAPTRALIAEELAEIVARQSSAYPGLDWPAVT RLVLDQRPLRSKGAGPCAFLPGEDRALRALPTVQDFIIRQTLANLRLPSTSADEPRPLTDEEHAKALALLSTARFVEWPALRR ALGLKRGVKFTAETERNGAKQAARGTAGNLTEAILAPLIPGWSGWDLDRKDRVFSDLWAARQDRSALLALIGDPRGPTRVTED ETAEAVADAIQIVLPTGRASLSAKAARAIAQAMAPGIGYDEAVTLALGLHHSHRPRQERLARLPYYAAALPDVGLDGDPVGPP PAEDDGAAAEAYYGRIGNISVHIALNETRKIVNALLHRHGPILRLVMVETTRELKAGADERKRMIAEQAERERENAEIDVELR KSDRWMANARERRQRVRLARRQNNLCPYTSTPIGHADLLGDAYDIDHVIPLARGGRDSLDNMVLCQSDANKTKGDKTPWEAFH DKPGWIAQRDDFLARLDPQTAKALAWRFADDAGERVARKSAEDEDQGFLPRQLTDTGYIARVALRYLSLVTNEPNAVVATNGR LTGLLRLAWDITPGPAPRDLLPTPRDALRDDTAARRFLDGLTPPPLAKAVEGAVQARLAALGRSRVADAGLADALGLTLASLG GGGKNRADHRHHFIDAAMIAVTTRGLINQINQASGAGRILDLRKWPRTNFEPPYPTFRAEVMKQWDHIHPSIRPAHRDGGSLH AATVFGVRNRPDARVLVQRKPVEKLFLDANAKPLPADKIAEIIDGFASPRMAKRFKALLARYQAAHPEVPPALAALAVARDPA FGPRGMTANTVIAGRSDGDGEDAGLITPFRANPKAAVRTMGNAVYEVWEIQVKGRPRWTHRVLTRFDRTQPAPPPPPENARLV MRLRRGDLVYWPLESGDRLFLVKKMAVDGRLALWPARLATGKATALYAQLSCPNINLNGDQGYCVQSAEGIRKEKIRTTSCTA LGRLRLSKKAT SEQ ID NO: 69 MKYTLGLDVGIASVGWAVIDKDNNKIIDLGVRCFDKAEESKTGESLATARRIARGMRRRISRRSQRLRLVKKLFVQYEIIKDS SEFNRIFDTSRDGWKDPWELRYNALSRILKPYELVQVLTHITKRRGFKSNRKEDLSTTKEGVVITSIKNNSEMLRTKNYRTIG EMIFMETPENSNKRNKVDEYIHTIAREDLLNEIKYIFSIQRKLGSPFVTEKLEHDFLNIWEFQRPFASGDSILSKVGKCTLLK EELRAPTSCYTSEYFGLLQSINNLVLVEDNNTLTLNNDQRAKIIEYAHFKNEIKYSEIRKLLDIEPEILFKAHNLTHKNPSGN NESKKFYEMKSYHKLKSTLPTDIWGKLHSNKESLDNLFYCLTVYKNDNEIKDYLQANNLDYLIEYIAKLPTFNKFKHLSLVAM KRIIPFMEKGYKYSDACNMAELDFTGSSKLEKCNKLTVEPIIENVTNPVVIRALTQARKVINAIIQKYGLPYMVNIELAREAG MTRQDRDNLKKEHENNRKAREKISDLIRQNGRVASGLDILKWRLWEDQGGRCAYSGKPIPVCDLLNDSLTQIDHIYPYSRSMD DSYMNKVLVLTDENQNKRSYTPYEVWGSTEKWEDFEARIYSMHLPQSKEKRLLNRNFITKDLDSFISRNLNDTRYISRFLKNY IESYLQFSNDSPKSCVVCVNGQCTAQLRSRWGLNKNREESDLHHALDAAVIACADRKIIKEITNYYNERENHNYKVKYPLPWH SFRQDLMETLAGVFISRAPRRKITGPAHDETIRSPKHFNKGLTSVKIPLTTVTLEKLETMVKNTKGGISDKAVYNVLKNRLIE HNNKPLKAFAEKIYKPLKNGTNGAIIRSIRVETPSYTGVFRNEGKGISDNSLMVRVDVFKKKDKYYLVPIYVAHMIKKELPSK AIVPLKPESQWELIDSTHEFLFSLYQNDYLVIKTKKGITEGYYRSCHRGTGSLSLMPHFANNKNVKIDIGVRTAISIEKYNVD ILGNKSIVKGEPRRGMEKYNSFKSN SEQ ID NO: 70 MIRTLGIDIGIASIGWAVIEGEYTDKGLENKEIVASGVRVFTKAENPKNKESLALPRTLARSARRRNARKKGRIQQVKHYLSK ALGLDLECFVQGEKLATLFQTSKDFLSPWELRERALYRVLDKEELARVILHIAKRRGYDDITYGVEDNDSGKIKKAIAENSKR IKEEQCKTIGEMMYKLYFQKSLNVRNKKESYNRCVGRSELREELKTIFQIQQELKSPWVNEELIYKLLGNPDAQSKQEREGLI FYQRPLKGFGDKIGKCSHIKKGENSPYRACKHAPSAEEFVALTKSINFLKNLTNRHGLCFSQEDMCVYLGKILQEAQKNEKGL TYSKLKLLLDLPSDFEFLGLDYSGKNPEKAVFLSLPSTFKLNKITQDRKTQDKIANILGANKDWEAILKELESLQLSKEQIQT IKDAKLNFSKHINLSLEALYHLLPLMNEGKRYDEGVEILQERGIFSKPQPKNRQLLPPLSELAKEESYFDIPNPVLRRALSEF RKVVNALLEKYGGFHYFHIELTRDVCKAKSARMQLEKINKKNKSENDAASQLLEVLGLPNTYNNRLKCKLWKQQEEYCLYSGE KITIDHLKDQRALQIDHAFPLSRSLDDSQSNKVLCLTSSNQEKSNKTPYEWLGSDEKKWDMYVGRVYSSNFSPSKKRKLTQKN FKERNEEDFLARNLVDTGYIGRVTKEYIKHSLSFLPLPDGKKEHIRIISGSMTSTMRSFWGVQEKNRDHHLHHAQDAIIIACI EPSMIQKYTTYLKDKETHRLKSHQKAQILREGDHKLSLRWPMSNFKDKIQESIQNIIPSHHVSHKVTGELHQETVRTKEFYYQ AFGGEEGVKKALKFGKIREINQGIVDNGAMVRVDIFKSKDKGKFYAVPIYTYDFAIGKLPNKAIVQGKKNGIIKDWLEMDENY EFCFSLFKNDCIKIQTKEMQEAVLAIYKSTNSAKATIELEHLSKYALKNEDEEKMFTDTDKEKNKTMTRESCGIQGLKVFQKV KLSVLGEVLEHKPRNRQNIALKTTPKHV SEQ ID NO: 71 MKYSIGLDIGIASVGWSVINKDKERIEDMGVRIFQKAENPKDGSSLASSRREKRGSRRRNRRKKHRLDRIKNILCESGLVKKN EIEKIYKNAYLKSPWELRAKSLEAKISNKEIAQILLHIAKRRGFKSFRKTDRNADDTGKLLSGIQENKKIMEEKGYLTIGDMV AKDPKFNTHVRNKAGSYLFSFSRKLLEDEVRKIQAKQKELGNTHFTDDVLEKYIEVFNSQRNFDEGPSKPSPYYSEIGQIAKM IGNCTFESSEKRTAKNTWSGERFVFLQKLNNFRIVGLSGKRPLTEEERDIVEKEVYLKKEVRYEKLRKILYLKEEERFGDLNY SKDEKQDKKTEKTKFISLIGNYTIKKLNLSEKLKSEIEEDKSKLDKIIEILTFNKSDKTIESNLKKLELSREDIEILLSEEFS GTLNLSLKAIKKILPYLEKGLSYNEACEKADYDYKNNGIKFKRGELLPVVDKDLIANPVVLRAISQTRKVVNAIIRKYGTPHT IHVEVARDLAKSYDDRQTIIKENKKRELENEKTKKFISEEFGIKNVKGKLLLKYRLYQEQEGRCAYSRKELSLSEVILDESMT DIDHIIPYSRSMDDSYSNKVLVLSGENRKKSNLLPKEYFDRQGRDWDTFVLNVKAMKIHPRKKSNLLKEKFTREDNKDWKSRA LNDTRYISRFVANYLENALEYRDDSPKKRVFMIPGQLTAQLRARWRLNKVRENGDLHHALDAAVVAVTDQKAINNISNISRYK ELKNCKDVIPSIEYHADEETGEVYFEEVKDTRFPMPWSGFDLELQKRLESENPREEFYNLLSDKRYLGWFNYEEGFIEKLRPV FVSRMPNRGVKGQAHQETIRSSKKISNQIAVSKKPLNSIKLKDLEKMQGRDTDRKLYEALKNRLEEYDDKPEKAFAEPFYKPT NSGKRGPLVRGIKVEEKQNVGVYVNGGQASNGSMVRIDVFRKNGKFYTVPIYVHQTLLKELPNRAINGKPYKDWDLIDGSFEF LYSFYPNDLIEIEFGKSKSIKNDNKLTKTEIPEVNLSEVLGYYRGMDTSTGAATIDTQDGKIQMRIGIKTVKNIKKYQVDVLG NVYKVKREKRQTF SEQ ID NO: 72 MSKKVSRRYEEQAQEICQRLGSRPYSIGLDLGVGSIGVAVAAYDPIKKQPSDLVFVSSRIFIPSTGAAERRQKRGQRNSLRHR ANRLKFLWKLLAERNLMLSYSEQDVPDPARLRFEDAVVRANPYELRLKGLNEQLTLSELGYALYHIANHRGSSSVRTFLDEEK SSDDKKLEEQQAMTEQLAKEKGISTFIEVLTAFNTNGLIGYRNSESVKSKGVPVPTRDIISNEIDVLLQTQKQFYQEILSDEY CDRIVSAILFENEKIVPEAGCCPYFPDEKKLPRCHFLNEERRLWEAINNARIKMPMQEGAAKRYQSASFSDEQRHILFHIARS GTDITPKLVQKEFPALKTSIIVLQGKEKAIQKIAGFRFRRLEEKSFWKRLSEEQKDDFFSAWTNTPDDKRLSKYLMKHLLLTE NEVVDALKTVSLIGDYGPIGKTATQLLMKHLEDGLTYTEALERGMETGEFQELSVWEQQSLLPYYGQILTGSTQALMGKYWHS AFKEKRDSEGFFKPNTNSDEEKYGRIANPVVHQTLNELRKLMNELITILGAKPQEITVELARELKVGAEKREDIIKQQTKQEK EAVLAYSKYCEPNNLDKRYIERFRLLEDQAFVCPYCLEHISVADIAAGRADVDHIFPRDDTADNSYGNKVVAHRQCNDIKGKR TPYAAFSNTSAWGPIMHYLDETPGMWRKRRKFETNEEEYAKYLQSKGFVSRFESDNSYIAKAAKEYLRCLFNPNNVTAVGSLK GMETSILRKAWNLQGIDDLLGSRHWSKDADTSPTMRKNRDDNRHHGLDAIVALYCSRSLVQMINTMSEQGKRAVEIEAMIPIP GYASEPNLSFEAQRELFRKKILEFMDLHAFVSMKTDNDANGALLKDTVYSILGADTQGEDLVFVVKKKIKDIGVKIGDYEEVA SAIRGRITDKQPKWYPMEMKDKIEQLQSKNEAALQKYKESLVQAAAVLEESNRKLIESGKKPIQLSEKTISKKALELVGGYYY LISNNKRTKTFVVKEPSNEVKGFAFDTGSNLCLDFYHDAQGKLCGEIIRKIQAMNPSYKPAYMKQGYSLYVRLYQGDVCELRA SDLTEAESNLAKTTHVRLPNAKPGRTFVIIITFTEMGSGYQIYFSNLAKSKKGQDTSFTLTTIKNYDVRKVQLSSAGLVRYVS PLLVDKIEKDEVALCGE SEQ ID NO: 73 MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRLERVKKLLEDYNLLDQSQIP QSTNPYAIRVKGLSEALSKDELVIALLHIAKRRGIHKIDVIDSNDDVGNELSTKEQLNKNSKLLKDKFVCQIQLERMNEGQVR GEKNRFKTADIIKEIIQLLNVQKNFHQLDENFINKYIELVEMRREYFEGPGKGSPYGWEGDPKAWYETLMGHCTYFPDELRSV KYAYSADLFNALNDLNNLVIQRDGLSKLEYHEKYHIIENVFKQKKKPTLKQIANEINVNPEDIKGYRITKSGKPQFTEFKLYH DLKSVLFDQSILENEDVLDQIAEILTIYQDKDSIKSKLTELDILLNEEDKENIAQLTGYTGTHRLSLKCIRLVLEEQWYSSRN QMEIFTHLNIKPKKINLTAANKIPKAMIDEFILSPVVKRTFGQAINLINKIIEKYGVPEDIIIELARENNSKDKQKFINEMQK KNENTRKRINEIIGKYGNQNAKRLVEKIRLHDEQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNSYHNKVLVKQSE NSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFEVQKEFINRNLVDTRYATRELTNYLKA YFSANNMNVKVKTINGSFTDYLRKVWKFKKERNHGYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIESKQLDIQVDSE DNYSEMFIIPKQVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQFDKSPEKFLMYQ HDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNKLGSHLDVTHQFKSSTKKLVKLSIKPYRF DVYLTDKGYKFITISYLDVLKKDNYYYIPEQKYDKLKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLP DIRYKEYCELNNIKGEPRIKKTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQLLFKRGN SEQ ID NO: 74 MIMKLEKWRLGLDLGTNSIGWSVFSLDKDNSVQDLIDMGVRIFSDGRDPKTKEPLAVARRTARSQRKLIYRRKLRRKQVFKFL QEQGLFPKTKEECMTLKSLNPYELRIKALDEKLEPYELGRALFNLAVRRGFKSNRKDGSREEVSEKKSPDEIKTQADMQTHLE KAIKENGCRTITEFLYKNQGENGGIRFAPGRMTYYPTRKMYEEEFNLIRSKQEKYYPQVDWDDIYKAIFYQRPLKPQQRGYCI YENDKERTFKAMPCSQKLRILQDIGNLAYYEGGSKKRVELNDNQDKVLYELLNSKDKVTFDQMRKALCLADSNSFNLEENRDF LIGNPTAVKMRSKNRFGKLWDEIPLEEQDLIIETIITADEDDAVYEVIKKYDLTQEQRDFIVKNTILQSGTSMLCKEVSEKLV KRLEEIADLKYHEAVESLGYKFADQTVEKYDLLPYYGKVLPGSTMEIDLSAFETNPEKHYGKISNPTVHVALNQTRVVVNALI KEYGKPSQIAIELSRDLKNNVEKKAEIARKQNQRAKENIAINDTISALYHTAFPGKSFYPNRNDRMKYRLWSELGLGNKCIYC GKGISGAELFTKEIEIEHILPFSRTLLDAESNLTVAHSSCNAFKAERSPFEAFGTNPSGYSWQEIIQRANQLKNTSKKNKFSP NAMDSFEKDSSFIARQLSDNQYIAKAALRYLKCLVENPSDVWTTNGSMTKLLRDKWEMDSILCRKFTEKEVALLGLKPEQIGN YKKNRFDHRHHAIDAVVIGLTDRSMVQKLATKNSHKGNRIEIPEFPILRSDLIEKVKNIVVSFKPDHGAEGKLSKETLLGKIK LHGKETFVCRENIVSLSEKNLDDIVDEIKSKVKDYVAKHKGQKIEAVLSDFSKENGIKKVRCVNRVQTPIEITSGKISRYLSP EDYFAAVIWEIPGEKKTFKAQYIRRNEVEKNSKGLNVVKPAVLENGKPHPAAKQVCLLHKDDYLEFSDKGKMYFCRIAGYAAT NNKLDIRPVYAVSYCADWINSTNETMLTGYWKPTPTQNWVSVNVLFDKQKARLVTVSPIGRVFRK SEQ ID NO: 75 MSSKAIDSLEQLDLFKPQEYTLGLDLGIKSIGWAILSGERIANAGVYLFETAEELNSTGNKLISKAAERGRKRRIRRMLDRKA RRGRHIRYLLEREGLPTDELEEVVVHQSNRTLWDVRAEAVERKLTKQELAAVLFHLVRHRGYFPNTKKLPPDDESDSADEEQG KINRATSRLREELKASDCKTIGQFLAQNRDRQRNREGDYSNLMARKLVFEEALQILAFQRKQGHELSKDFEKTYLDVLMGQRS GRSPKLGNCSLIPSELRAPSSAPSTEWFKFLQNLGNLQISNAYREEWSIDAPRRAQIIDACSQRSTSSYWQIRRDFQIPDEYR FNLVNYERRDPDVDLQEYLQQQERKTLANFRNWKQLEKIIGTGHPIQTLDEAARLITLIKDDEKLSDQLADLLPEASDKAITQ LCELDFTTAAKISLEAMYRILPHMNQGMGFFDACQQESLPEIGVPPAGDRVPPFDEMYNPVVNRVLSQSRKLINAVIDEYGMP AKIRVELARDLGKGRELRERIKLDQLDKSKQNDQRAEDFRAEFQQAPRGDQSLRYRLWKEQNCTCPYSGRMIPVNSVLSEDTQ IDHILPISQSFDNSLSNKVLCFTEENAQKSNRTPFEYLDAADFQRLEAISGNWPEAKRNKLLHKSFGKVAEEWKSRALNDTRY LTSALADHLRHHLPDSKIQTVNGRITGYLRKQWGLEKDRDKHTHHAVDAIVVACTTPAIVQQVTLYHQDIRRYKKLGEKRPTP WPETFRQDVLDVEEEIFITRQPKKVSGGIQTKDTLRKHRSKPDRQRVALTKVKLADLERLVEKDASNRNLYEHLKQCLEESGD QPTKAFKAPFYMPSGPEAKQRPILSKVTLLREKPEPPKQLTELSGGRRYDSMAQGRLDIYRYKPGGKRKDEYRVVLQRMIDLM RGEENVHVFQKGVPYDQGPEIEQNYTFLFSLYFDDLVEFQRSADSEVIRGYYRTFNIANGQLKISTYLEGRQDFDFFGANRLA HFAKVQVNLLGKVIK SEQ ID NO: 76 MRSLRYRLALDLGSTSLGWALFRLDACNRPTAVIKAGVRIFSDGRNPKDGSSLAVTRRAARAMRRRRDRLLKRKTRMQAKLVE HGFFPADAGKRKALEQLNPYALRAKGLQEALLPGEFARALFHINQRRGFKSNRKTDKKDNDSGVLKKAIGQLRQQMAEQGSRT VGEYLWTRLQQGQGVRARYREKPYTTEEGKKRIDKSYDLYIDRAMIEQEFDALWAAQAAFNPTLFHEAARADLKDTLLHQRPL RPVKPGRCTLLPEEERAPLALPSTQRFRIHQEVNHLRLLDENLREVALTLAQRDAVVTALETKAKLSFEQIRKLLKLSGSVQF NLEDAKRTELKGNATSAALARKELFGAAWSGFDEALQDEIVWQLVTEEGEGALIAWLQTHTGVDEARAQAIVDVSLPEGYGNL SRKALARIVPALRAAVITYDKAVQAAGFDHHSQLGFEYDASEVEDLVHPETGEIRSVFKQLPYYGKALQRHVAFGSGKPEDPD EKRYGKIANPTVHIGLNQVRMVVNALIRRYGRPTEVVIELARDLKQSREQKVEAQRRQADNQRRNARIRRSIAEVLGIGEERV RGSDIQKWICWEELSFDAADRRCPYSGVQISAAMLLSDEVEVEHILPFSKTLDDSLNNRTVAMRQANRIKRNRTPWDARAEFE AQGWSYEDILQRAERMPLRKRYRFAPDGYERWLGDDKDFLARALNDTRYLSRVAAEYLRLVCPGTRVIPGQLTALLRGKFGLN DVLGLDGEKNRNDHRHHAVDACVIGVTDQGLMQRFATASAQARGDGLTRLVDGMPMPWPTYRDHVERAVRHIWVSHRPDHGFE GAMMEETSYGIRKDGSIKQRRKADGSAGREISNLIRIHEATQPLRHGVSADGQPLAYKGYVGGSNYCIEITVNDKGKWEGEVI STFRAYGVVRAGGMGRLRNPHEGQNGRKLIMRLVIGDSVRLEVDGAERTMRIVKISGSNGQIFMAPIHEANVDARNTDKQDAF TYTSKYAGSLQKAKTRRVTISPIGEVRDPGFKG SEQ ID NO: 77 MARPAFRAPRREHVNGWTPDPHRISKPFFILVSWHLLSRVVIDSSSGCFPGTSRDHTDKFAEWECAVQPYRLSFDLGTNSIGW GLLNLDRQGKPREIRALGSRIFSDGRDPQDKASLAVARRLARQMRRRRDRYLTRRTRLMGALVRFGLMPADPAARKRLEVAVD PYLARERATRERLEPFEIGRALFHLNQRRGYKPVRTATKPDEEAGKVKEAVERLEAAIAAAGAPTLGAWFAWRKTRGETLRAR LAGKGKEAAYPFYPARRMLEAEFDTLWAEQARHHPDLLTAEAREILRHRIFHQRPLKPPPVGRCTLYPDDGRAPRALPSAQRL RLFQELASLRVIHLDLSERPLTPAERDRIVAFVQGRPPKAGRKPGKVQKSVPFEKLRGLLELPPGTGFSLESDKRPELLGDET GARIAPAFGPGWTALPLEEQDALVELLLTEAEPERAIAALTARWALDEATAAKLAGATLPDFHGRYGRRAVAELLPVLERETR GDPDGRVRPIRLDEAVKLLRGGKDHSDFSREGALLDALPYYGAVLERHVAFGTGNPADPEEKRVGRVANPTVHIALNQLRHLV NAILARHGRPEEIVIELARDLKRSAEDRRREDKRQADNQKRNEERKRLILSLGERPTPRNLLKLRLWEEQGPVENRRCPYSGE TISMRMLLSEQVDIDHILPFSVSLDDSAANKVVCLREANRIKRNRSPWEAFGHDSERWAGILARAEALPKNKRWRFAPDALEK LEGEGGLRARHLNDTRHLSRLAVEYLRCVCPKVRVSPGRLTALLRRRWGIDAILAEADGPPPEVPAETLDPSPAEKNRADHRH HALDAVVIGCIDRSMVQRVQLAAASAEREAAAREDNIRRVLEGFKEEPWDGFRAELERRARTIVVSHRPEHGIGGALHKETAY GPVDPPEEGFNLVVRKPIDGLSKDEINSVRDPRLRRALIDRLAIRRRDANDPATALAKAAEDLAAQPASRGIRRVRVLKKESN PIRVEHGGNPSGPRSGGPFHKLLLAGEVHHVDVALRADGRRWVGHWVTLFEAHGGRGADGAAAPPRLGDGERFLMRLHKGDCL KLEHKGRVRVMQVVKLEPSSNSVVVVEPHQVKTDRSKHVKISCDQLRARGARRVTVDPLGRVRVHAPGARVGIGGDAGRTAME PAEDIS SEQ ID NO: 78 MKRTSLRAYRLGVDLGANSLGWFVVWLDDHGQPEGLGPGGVRIFPDGRNPQSKQSNAAGRRLARSARRRRDRYLQRRGKLMGL LVKHGLMPADEPARKRLECLDPYGLRAKALDEVLPLHHVGRALFHLNQRRGLFANRAIEQGDKDASAIKAAAGRLQTSMQACG ARTLGEFLNRRHQLRATVRARSPVGGDVQARYEFYPTRAMVDAEFEAIWAAQAPHHPTMTAEAHDTIREAIFSQRAMKRPSIG KCSLDPATSQDDVDGFRCAWSHPLAQRFRIWQDVRNLAVVETGPTSSRLGKEDQDKVARALLQTDQLSFDEIRGLLGLPSDAR FNLESDRRDHLKGDATGAILSARRHFGPAWHDRSLDRQIDIVALLESALDEAAIIASLGTTHSLDEAAAQRALSALLPDGYCR LGLRAIKRVLPLMEAGRTYAEAASAAGYDHALLPGGKLSPTGYLPYYGQWLQNDVVGSDDERDTNERRWGRLPNPTVHIGIGQ LRRVVNELIRWHGPPAEITVELTRDLKLSPRRLAELEREQAENQRKNDKRTSLLRKLGLPASTHNLLKLRLWDEQGDVASECP YTGEAIGLERLVSDDVDIDHLIPFSISWDDSAANKVVCMRYANREKGNRTPFEAFGHRQGRPYDWADIAERAARLPRGKRWRF GPGARAQFEELGDFQARLLNETSWLARVAKQYLAAVTHPHRIHVLPGRLTALLRATWELNDLLPGSDDRAAKSRKDHRHHAID ALVAALTDQALLRRMANAHDDTRRKIEVLLPWPTFRIDLETRLKAMLVSHKPDHGLQARLHEDTAYGTVEHPETEDGANLVYR KTFVDISEKEIDRIRDRRLRDLVRAHVAGERQQGKTLKAAVLSFAQRRDIAGHPNGIRHVRLTKSIKPDYLVPIRDKAGRIYK SYNAGENAFVDILQAESGRWIARATTVFQANQANESHDAPAAQPIMRVFKGDMLRIDHAGAEKFVKIVRLSPSNNLLYLVEHH QAGVFQTRHDDPEDSFRWLFASFDKLREWNAELVRIDTLGQPWRRKRGLETGSEDATRIGWTRPKKWP SEQ ID NO: 79 MERIFGFDIGTTSIGFSVIDYSSTQSAGNIQRLGVRIFPEARDPDGTPLNQQRRQKRMMRRQLRRRRIRRKALNETLHEAGFL PAYGSADWPVVMADEPYELRRRGLEEGLSAYEFGRAIYHLAQHRHFKGRELEESDTPDPDVDDEKEAANERAATLKALKNEQT TLGAWLARRPPSDRKRGIHAHRNVVAEEFERLWEVQSKFHPALKSEEMRARISDTIFAQRPVFWRKNTLGECRFMPGEPLCPK GSWLSQQRRMLEKLNNLAIAGGNARPLDAEERDAILSKLQQQASMSWPGVRSALKALYKQRGEPGAEKSLKFNLELGGESKLL GNALEAKLADMFGPDWPAHPRKQEIRHAVHERLWAADYGETPDKKRVIILSEKDRKAHREAAANSFVADFGITGEQAAQLQAL KLPTGWEPYSIPALNLFLAELEKGERFGALVNGPDWEGWRRTNFPHRNQPTGEILDKLPSPASKEERERISQLRNPTVVRTQN ELRKVVNNLIGLYGKPDRIRIEVGRDVGKSKREREEIQSGIRRNEKQRKKATEDLIKNGIANPSRDDVEKWILWKEGQERCPY TGDQIGFNALFREGRYEVEHIWPRSRSFDNSPRNKTLCRKDVNIEKGNRMPFEAFGHDEDRWSAIQIRLQGMVSAKGGTGMSP GKVKRFLAKTMPEDFAARQLNDTRYAAKQILAQLKRLWPDMGPEAPVKVEAVTGQVTAQLRKLWTLNNILADDGEKTRADHRH HAIDALTVACTHPGMTNKLSRYWQLRDDPRAEKPALTPPWDTIRADAEKAVSEIVVSHRVRKKVSGPLHKETTYGDTGTDIKT KSGTYRQFVTRKKIESLSKGELDEIRDPRIKEIVAAHVAGRGGDPKKAFPPYPCVSPGGPEIRKVRLTSKQQLNLMAQTGNGY ADLGSNHHIAIYRLPDGKADFEIVSLFDASRRLAQRNPIVQRTRADGASFVMSLAAGEAIMIPEGSKKGIWIVQGVWASGQVV LERDTDADHSTTTRPMPNPILKDDAKKVSIDPIGRVRPSND SEQ ID NO: 80 MNKRILGLDTGTNSLGWAVVDWDEHAQSYELIKYGDVIFQEGVKIEKGIESSKAAERSGYKAIRKQYFRRRLRKIQVLKVLVK YHLCPYLSDDDLRQWHLQKQYPKSDELMLWQRTSDEEGKNPYYDRHRCLHEKLDLTVEADRYTLGRALYHLTQRRGFLSNRLD TSADNKEDGVVKSGISQLSTEMEEAGCEYLGDYFYKLYDAQGNKVRIRQRYTDRNKHYQHEFDAICEKQELSSELIEDLQRAI FFQLPLKSQRHGVGRCTFERGKPRCADSHPDYEEFRMLCFVNNIQVKGPHDLELRPLTYEEREKIEPLFFRKSKPNFDFEDIA KALAGKKNYAWIHDKEERAYKFNYRMTQGVPGCPTIAQLKSIFGDDWKTGIAETYTLIQKKNGSKSLQEMVDDVWNVLYSFSS VEKLKEFAHHKLQLDEESAEKFAKIKLSHSFAALSLKAIRKFLPFLRKGMYYTHASFFANIPTIVGKEIWNKEQNRKYIMENV GELVFNYQPKHREVQGTIEMLIKDFLANNFELPAGATDKLYHPSMIETYPNAQRNEFGILQLGSPRTNAIRNPMAMRSLHILR RVVNQLLKESIIDENTEVHVEYARELNDANKRRAIADRQKEQDKQHKKYGDEIRKLYKEETGKDIEPTQTDVLKFQLWEEQNH HCLYTGEQIGITDFIGSNPKFDIEHTIPQSVGGDSTQMNLTLCDNRFNREVKKAKLPTELANHEEILTRIEPWKNKYEQLVKE RDKQRTFAGMDKAVKDIRIQKRHKLQMEIDYWRGKYERFTMTEVPEGFSRRQGTGIGLISRYAGLYLKSLFHQADSRNKSNVY VVKGVATAEFRKMWGLQSEYEKKCRDNHSHHCMDAITIACIGKREYDLMAEYYRMEETFKQGRGSKPKFSKPWATFTEDVLNI YKNLLVVHDTPNNMPKHTKKYVQTSIGKVLAQGDTARGSLHLDTYYGAIERDGEIRYVVRRPLSSFTKPEELENIVDETVKRT IKEAIADKNFKQAIAEPIYMNEEKGILIKKVRCFAKSVKQPINIRQHRDLSKKEYKQQYHVMNENNYLLAIYEGLVKNKVVRE FEIVSYIEAAKYYKRSQDRNIFSSIVPTHSTKYGLPLKTKLLMGQLVLMFEENPDEIQVDNTKDLVKRLYKVVGIEKDGRIKF KYHQEARKEGLPIFSTPYKNNDDYAPIFRQSINNINILVDGIDFTIDILGKVTLKE SEQ ID NO: 81 MNYKMGLDIGIASVGWAVINLDLKRIEDLGVRIFDKAEHPQNGESLALPRRIARSARRRLRRRKHRLERIRRLLVSENVLTKE EMNLLFKQKKQIDVWQLRVDALERKLNNDELARVLLHLAKRRGFKSNRKSERNSKESSEFLKNIEENQSILAQYRSVGEMIVK DSKFAYHKRNKLDSYSNMIARDDLEREIKLIFEKQREFNNPVCTERLEEKYLNIWSSQRPFASKEDIEKKVGFCTFEPKEKRA PKATYTFQSFIVWEHINKLRLVSPDETRALTEIERNLLYKQAFSKNKMTYYDIRKLLNLSDDIHFKGLLYDPKSSLKQIENIR FLELDSYHKIRKCIENVYGKDGIRMFNETDIDTFGYALTIFKDDEDIVAYLQNEYITKNGKRVSNLANKVYDKSLIDELLNLS FSKFAHLSMKAIRNILPYMEQGEIYSKACELAGYNFTGPKKKEKALLLPVIPNIANPVVMRALTQSRKVVNAIIKKYGSPVSI HIELARDLSHSFDERKKIQKDQTENRKKNETAIKQLIEYELTKNPTGLDIVKFKLWSEQQGRCMYSLKPIELERLLEPGYVEV DHILPYSRSLDDSYANKVLVLTKENREKGNHTPVEYLGLGSERWKKFEKFVLANKQFSKKKKQNLLRLRYEETEEKEFKERNL NDTRYISKFFANFIKEHLKFADGDGGQKVYTINGKITAHLRSRWDFNKNREESDLHHAVDAVIVACATQGMIKKITEFYKARE QNKESAKKKEPIFPQPWPHFADELKARLSKFPQESIEAFALGNYDRKKLESLRPVFVSRMPKRSVTGAAHQETLRRCVGIDEQ SGKIQTAVKTKLSDIKLDKDGHFPMYQKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGEPGPVIRTVKIIDTKNKVV HLDGSKTVAYNSNIVRTDVFEKDGKYYCVPVYTMDIMKGTLPNKAIEANKPYSEWKEMTEEYTFQFSLFPNDLVRIVLPREKT IKTSTNEEIIIKDIFAYYKTIDSATGGLELISHDRNFSLRGVGSKTLKRFEKYQVDVLGNIHKVKGEKRVGLAAPTNQKKGKT VDSLQSVSD SEQ ID NO: 82 MRRLGLDLGTNSIGWCLLDLGDDGEPVSIFRTGARIFSDGRDPKSLGSLKATRREARLTRRRRDRFIQRQKNLINALVKYGLM PADEIQRQALAYKDPYPIRKKALDEAIDPYEMGRAIFHINQRRGFKSNRKSADNEAGVVKQSIADLEMKLGEAGARTIGEFLA DRQATNDTVRARRLSGTNALYEFYPDRYMLEQEFDTLWAKQAAFNPSLYIEAARERLKEIVFFQRKLKPQEVGRCIFLSDEDR ISKALPSFQRFRIYQELSNLAWIDHDGVAHRITASLALRDHLFDELEHKKKLTFKAMRAILRKQGVVDYPVGFNLESDNRDHL IGNLTSCIMRDAKKMIGSAWDRLDEEEQDSFILMLQDDQKGDDEVRSILTQQYGLSDDVAEDCLDVRLPDGHGSLSKKAIDRI LPVLRDQGLIYYDAVKEAGLGEANLYDPYAALSDKLDYYGKALAGHVMGASGKFEDSDEKRYGTISNPTVHIALNQVRAVVNE LIRLHGKPDEVVIEIGRDLPMGADGKRELERFQKEGRAKNERARDELKKLGHIDSRESRQKFQLWEQLAKEPVDRCCPFTGKM MSISDLFSDKVEIEHLLPFSLTLDDSMANKTVCFRQANRDKGNRAPFDAFGNSPAGYDWQEILGRSQNLPYAKRWRFLPDAMK RFEADGGFLERQLNDTRYISRYTTEYISTIIPKNKIWVVTGRLTSLLRGFWGLNSILRGHNTDDGTPAKKSRDDHRHHAIDAI VVGMTSRGLLQKVSKAARRSEDLDLTRLFEGRIDPWDGFRDEVKKHIDAIIVSHRPRKKSQGALHNDTAYGIVEHAENGASTV VHRVPITSLGKQSDIEKVRDPLIKSALLNETAGLSGKSFENAVQKWCADNSIKSLRIVETVSIIPITDKEGVAYKGYKGDGNA YMDIYQDPTSSKWKGEIVSRFDANQKGFIPSWQSQFPTARLIMRLRINDLLKLQDGEIEEIYRVQRLSGSKILMAPHTEANVD ARDRDKNDTFKLTSKSPGKLQSASARKVHISPTGLIREG SEQ ID NO: 83 MKNILGLDLGLSSIGWSVIRENSEEQELVAMGSRVVSLTAAELSSFTQGNGVSINSQRTQKRTQRKGYDRYQLRRTLLRNKLD TLGMLPDDSLSYLPKLQLWGLRAKAVTQRIELNELGRVLLHLNQKRGYKSIKSDFSGDKKITDYVKTVKTRYDELKEMRLTIG ELFFRRLTENAFFRCKEQVYPRQAYVEEFDCIMNCQRKFYPDILTDETIRCIRDEIIYYQRPLKSCKYLVSRCEFEKRFYLNA AGKKTEAGPKVSPRTSPLFQVCRLWESINNIVVKDRRNEIVFISAEQRAALFDFLNTHEKLKGSDLLKLLGLSKTYGYRLGEQ FKTGIQGNKTRVEIERALGNYPDKKRLLQFNLQEESSSMVNTETGEIIPMISLSFEQEPLYRLWHVLYSIDDREQLQSVLRQK FGIDDDEVLERLSAIDLVKAGFGNKSSKAIRRILPFLQLGMNYAEACEAAGYNHSNNYTKAENEARALLDRLPAIKKNELRQP VVEKILNQMVNVVNALMEKYGRFDEIRVELARELKQSKEERSNTYKSINKNQRENEQIAKRIVEYGVPTRSRIQKYKMWEESK HCCIYCGQPVDVGDFLRGFDVEVEHIIPKSLYFDDSFANKVCSCRSCNKEKNNRTAYDYMKSKGEKALSDYVERVNTMYTNNQ ISKTKWQNLLTPVDKISIDFIDRQLRESQYIARKAKEILTSICYNVTATSGSVTSFLRHVWGWDTVLHDLNFDRYKKVGLTEV IEVNHRGSVIRREQIKDWSKRFDHRHHAIDALTIACTKQAYIQRLNNLRAEEGPDFNKMSLERYIQSQPHFSVAQVREAVDRI LVSFRAGKRAVTPGKRYIRKNRKRISVQSVLIPRGALSEESVYGVIHVWEKDEQGHVIQKQRAVMKYPITSINREMLDKEKVV DKRIHRILSGRLAQYNDNPKEAFAKPVYIDKECRIPIRTVRCFAKPAINTLVPLKKDDKGNPVAWVNPGNNHHVAIYRDEDGK YKERTVTFWEAVDRCRVGIPAIVTQPDTIWDNILQRNDISENVLESLPDVKWQFVLSLQQNEMFILGMNEEDYRYAMDQQDYA LLNKYLYRVQKLSKSDYSFRYHTETSVEDKYDGKPNLKLSMQMGKLKRVSIKSLLGLNPHKVHISVLGEIKEIS SEQ ID NO: 84 MAEKQHRWGLDIGTNSIGWAVIALIEGRPAGLVATGSRIFSDGRNPKDGSSLAVERRGPRQMRRRRDRYLRRRDRFMQALINV GLMPGDAAARKALVTENPYVLRQRGLDQALTLPEFGRALFHLNQRRGFQSNRKTDRATAKESGKVKNAIAAFRAGMGNARTVG EALARRLEDGRPVRARMVGQGKDEHYELYIAREWIAQEFDALWASQQRFHAEVLADAARDRLRAILLFQRKLLPVPVGKCFLE PNQPRVAAALPSAQRFRLMQELNHLRVMTLADKRERPLSFQERNDLLAQLVARPKCGFDMLRKIVFGANKEAYRFTIESERRK ELKGCDTAAKLAKVNALGTRWQALSLDEQDRLVCLLLDGENDAVLADALREHYGLTDAQIDTLLGLSFEDGHMRLGRSALLRV LDALESGRDEQGLPLSYDKAVVAAGYPAHTADLENGERDALPYYGELLWRYTQDAPTAKNDAERKFGKIANPTVHIGLNQLRK LVNALIQRYGKPAQIVVELARNLKAGLEEKERIKKQQTANLERNERIRQKLQDAGVPDNRENRLRMRLFEELGQGNGLGTPCI YSGRQISLQRLFSNDVQVDHILPFSKTLDDSFANKVLAQHDANRYKGNRGPFEAFGANRDGYAWDDIRARAAVLPRNKRNRFA ETAMQDWLHNETDFLARQLTDTAYLSRVARQYLTAICSKDDVYVSPGRLTAMLRAKWGLNRVLDGVMEEQGRPAVKNRDDHRH HAIDAVVIGATDRAMLQQVATLAARAREQDAERLIGDMPTPWPNFLEDVRAAVARCVVSHKPDHGPEGGLHNDTAYGIVAGPF EDGRYRVRHRVSLFDLKPGDLSNVRCDAPLQAELEPIFEQDDARAREVALTALAERYRQRKVWLEELMSVLPIRPRGEDGKTL PDSAPYKAYKGDSNYCYELFINERGRWDGELISTFRANQAAYRRFRNDPARFRRYTAGGRPLLMRLCINDYIAVGTAAERTIF RVVKMSENKITLAEHFEGGTLKQRDADKDDPFKYLTKSPGALRDLGARRIFVDLIGRVLDPGIKGD SEQ ID NO: 86 MIERILGVDLGISSLGWAIVEYDKDDEAANRIIDCGVRLFTAAETPKKKESPNKARREARGIRRVLNRRRVRMNMIKKLFLRA GLIQDVDLDGEGGMFYSKANRADVWELRHDGLYRLLKGDELARVLIHIAKHRGYKFIGDDEADEESGKVKKAGVVLRQNFEAA GCRTVGEWLWRERGANGKKRNKHGDYEISIHRDLLVEEVEAIFVAQQEMRSTIATDALKAAYREIAFFVRPMQRIEKMVGHCT YFPEERRAPKSAPTAEKFIAISKFFSTVIIDNEGWEQKIIERKTLEELLDFAVSREKVEFRHLRKFLDLSDNEIFKGLHYKGK PKTAKKREATLFDPNEPTELEFDKVEAEKKAWISLRGAAKLREALGNEFYGRFVALGKHADEATKILTYYKDEGQKRRELTKL PLEAEMVERLVKIGFSDFLKLSLKAIRDILFAMESGARYDEAVLMLGVPHKEKSAILPPLNKTDIDILNPTVIRAFAQFRKVA NALVRKYGAFDRVHFELAREINTKGEIEDIKESQRKNEKERKEAADWIAETSFQVPLTRKNILKKRLYIQQDGRCAYTGDVIE LERLFDEGYCEIDHILPRSRSADDSFANKVLCLARANQQKTDRTPYEWFGHDAARWNAFETRTSAPSNRVRTGKGKIDRLLKK NFDENSEMAFKDRNLNDTRYMARAIKTYCEQYWVFKNSHTKAPVQVRSGKLTSVLRYQWGLESKDRESHTHHAVDAIIIAFST QGMVQKLSEYYRFKETHREKERPKLAVPLANFRDAVEEATRIENTETVKEGVEVKRLLISRPPRARVTGQAHEQTAKPYPRIK QVKNKKKWRLAPIDEEKFESFKADRVASANQKNFYETSTIPRVDVYHKKGKFHLVPIYLHEMVLNELPNLSLGTNPEAMDENF FKFSIFKDDLISIQTQGTPKKPAKIIMGYFKNMHGANMVLSSINNSPCEGFTCTPVSMDKKHKDKCKLCPEENRIAGRCLQGF LDYWSQEGLRPPRKEFECDQGVKFALDVKKYQIDPLGYYYEVKQEKRLGTIPQMRSAKKLVKK SEQ ID NO: 87 MNNSIKSKPEVTIGLDLGVGSVGWAIVDNETNIIHHLGSRLFSQAKTAEDRRSFRGVRRLIRRRKYKLKRFVNLIWKYNSYFGFKNKEDILNNY QEQQKLHNTVLNLKSEALNAKIDPKALSWILHDYLKNRGHFYEDNRDFNVYPTKELAKYFDKYGYYKGIIDSKEDNDNKLEEELTKYKFSNKHW LEEVKKVLSNQTGLPEKFKEEYESLFSYVRNYSEGPGSINSVSPYGIYHLDEKEGKVVQKYNNIWDKTIGKCNIFPDEYRAPKNSPIAMIFNEI NELSTIRSYSIYLTGWFINQEFKKAYLNKLLDLLIKTNGEKPIDARQFKKLREETIAESIGKETLKDVENEEKLEKEDHKWKLKGLKLNTNGKI QYNDLSSLAKFVHKLKQHLKLDFLLEDQYATLDKINFLQSLFVYLGKHLRYSNRVDSANLKEFSDSNKLFERILQKQKDGLFKLFEQTDKDDEK ILAQTHSLSTKAMLLAITRMTNLDNDEDNQKNNDKGWNFEAIKNFDQKFIDITKKNNNLSLKQNKRYLDDRFINDAILSPGVKRILREATKVFN AILKQFSEEYDVTKVVIELARELSEEKELENTKNYKKLIKKNGDKISEGLKALGISEDEIKDILKSPTKSYKFLLWLQQDHIDPYSLKEIAFDD IFTKTEKFEIDHIIPYSISFDDSSSNKLLVLAESNQAKSNQTPYEFISSGNAGIKWEDYEAYCRKFKDGDSSLLDSTQRSKKFAKMMKTDTSSK YDIGFLARNLNDTRYATIVFRDALEDYANNHLVEDKPMFKVVCINGSVTSFLRKNFDDSSYAKKDRDKNIHHAVDASIISIFSNETKTLFNQLT QFADYKLFKNTDGSWKKIDPKTGVVTEVTDENWKQIRVRNQVSEIAKVIEKYIQDSNIERKARYSRKIENKTNISLFNDTVYSAKKVGYEDQIK RKNLKTLDIHESAKENKNSKVKRQFVYRKLVNVSLLNNDKLADLFAEKEDILMYRANPWVINLAEQIFNEYTENKKIKSQNVFEKYMLDLTKEF PEKFSEFLVKSMLRNKTAIIYDDKKNIVHRIKRLKMLSSELKENKLSNVIIRSKNQSGTKLSYQDTINSLALMIMRSIDPTAKKQYIRVPLNTL NLHLGDHDFDLHNMDAYLKKPKFVKYLKANEIGDEYKPWRVLTSGTLLIHKKDKKLMYISSFQNLNDVIEIKNLIETEYKENDDSDSKKKKKAN RFLMTLSTILNDYILLDAKDNFDILGLSKNRIDEILNSKLGLDKIVK SEQ ID NO: 88 MGGSEVGTVPVTWRLGVDVGERSIGLAAVSYEEDKPKEILAAVSWIHDGGVGDERSGASRLALRGMARRARRLRRFRRARLRDLDMLLSELGWT PLPDKNVSPVDAWLARKRLAEEYVVDETERRRLLGYAVSHMARHRGWRNPWTTIKDLKNLPQPSDSWERTRESLEARYSVSLEPGTVGQWAGYL LQRAPGIRLNPTQQSAGRRAELSNATAFETRLRQEDVLWELRCIADVQGLPEDVVSNVIDAVFCQKRPSVPAERIGRDPLDPSQLRASRACLEF QEYRIVAAVANLRIRDGSGSRPLSLEERNAVIEALLAQTERSLTWSDIALEILKLPNESDLTSVPEEDGPSSLAYSQFAPFDETSARIAEFIAK NRRKIPTFAQWWQEQDRTSRSDLVAALADNSIAGEEEQELLVHLPDAELEALEGLALPSGRVAYSRLTLSGLTRVMRDDGVDVHNARKTCFGVD DNWRPPLPALHEATGHPVVDRNLAILRKFLSSATMRWGPPQSIVVELARGASESRERQAEEEAARRAHRKANDRIRAELRASGLSDPSPADLVR ARLLELYDCHCMYCGAPISWENSELDHIVPRTDGGSNRHENLAITCGACNKEKGRRPFASWAETSNRVQLRDVIDRVQKLKYSGNMYWTRDEFS RYKKSVVARLKRRTSDPEVIQSIESTGYAAVALRDRLLSYGEKNGVAQVAVFRGGVTAEARRWLDISIERLFSRVAIFAQSTSTKRLDRRHHAV DAVVLTTLTPGVAKTLADARSRRVSAEFWRRPSDVNRHSTEEPQSPAYRQWKESCSGLGDLLISTAARDSIAVAAPLRLRPTGALHEETLRAFS EHTVGAAWKGAELRRIVEPEVYAAFLALTDPGGRFLKVSPSEDVLPADENRHIVLSDRVLGPRDRVKLFPDDRGSIRVRGGAAYIASFHHARVF RWGSSHSPSFALLRVSLADLAVAGLLRDGVDVFTAELPPWTPAWRYASIALVKAVESGDAKQVGWLVPGDELDFGPEGVTTAAGDLSMFLKYFP ERHWVVTGFEDDKRINLKPAFLSAEQAEVLRTERSDRPDTLTEAGEILAQFFPRCWRATVAKVLCHPGLTVIRRTALGQPRWRRGHLPYSWRPW SADPWSGGTP SEQ ID NO: 89 MHNKKNITIGFDLGIASIGWAIIDSTTSKILDWGTRTFEERKTANERRAFRSTRRNIRRKAYRNQRFINLILKYKDLFELKNISDIQRANKKDT ENYEKIISFFTEIYKKCAAKHSNILEVKVKALDSKIEKLDLIWILHDYLENRGFFYDLEEENVADKYEGIEHPSILLYDFFKKNGFFKSNSSIP KDLGGYSFSNLQWVNEIKKLFEVQEINPEFSEKFLNLFTSVRDYAKGPGSEHSASEYGIFQKDEKGKVFKKYDNIWDKTIGKCSFFVEENRSPV NYPSYEIFNLLNQLINLSTDLKTTNKKIWQLSSNDRNELLDELLKVKEKAKIISISLKKNEIKKIILKDFGFEKSDIDDQDTIEGRKIIKEEPT TKLEVTKHLLATIYSHSSDSNWININNILEFLPYLDAICIILDREKSRGQDEVLKKLTEKNIFEVLKIDREKQLDFVKSIFSNTKFNFKKIGNF SLKAIREFLPKMFEQNKNSEYLKWKDEEIRRKWEEQKSKLGKTDKKTKYLNPRIFQDEIISPGTKNTFEQAVLVLNQIIKKYSKENIIDAIIIE SPREKNDKKTIEEIKKRNKKGKGKTLEKLFQILNLENKGYKLSDLETKPAKLLDRLRFYHQQDGIDLYTLDKINIDQLINGSQKYEIEHIIPYS MSYDNSQANKILTEKAENLKKGKLIASEYIKRNGDEFYNKYYEKAKELFINKYKKNKKLDSYVDLDEDSAKNRFRFLTLQDYDEFQVEFLARNL NDTRYSTKLFYHALVEHFENNEFFTYIDENSSKHKVKISTIKGHVTKYFRAKPVQKNNGPNENLNNNKPEKIEKNRENNEHHAVDAAIVAIIGN KNPQIANLLTLADNKTDKKFLLHDENYKENIETGELVKIPKFEVDKLAKVEDLKKIIQEKYEEAKKHTAIKFSRKTRTILNGGLSDETLYGFKY DEKEDKYFKIIKKKLVTSKNEELKKYFENPFGKKADGKSEYTVLMAQSHLSEFNKLKEIFEKYNGFSNKTGNAFVEYMNDLALKEPTLKAEIES AKSVEKLLYYNFKPSDQFTYHDNINNKSFKRFYKNIRIIEYKSIPIKFKILSKHDGGKSFKDTLFSLYSLVYKVYENGKESYKSIPVTSQMRNF GIDEFDFLDENLYNKEKLDIYKSDFAKPIPVNCKPVFVLKKGSILKKKSLDIDDFKETKETEEGNYYFISTISKRFNRDTAYGLKPLKLSVVKP VAEPSTNPIFKEYIPIHLDELGNEYPVKIKEHTDDEKLMCTIK DNA2 endonuclease (CCDS 44415.2) SEQ ID NO: 85 MEQLNELELLMEKSFWEEAELPAELFQKKVVASFPRTVLSTGMDNRYLVLAVNTVQNKEGNCEKRLVITASQSLENKELCILRNDWCSVPVEPG DIIHLEGDCTSDTWIIDKDFGYLILYPDMLISGTSIASSIRCMRRAVLSETFRSSDPATRQMLIGTVLHEVFQKAINNSFAPEKLQELAFQTIQ EIRHLKEMYRLNLSQDEIKQEVEDYLPSFCKWAGDFMHKNTSTDFPQMQLSLPSDNSKDNSTCNIEVVKPMDIEESIWSPRFGLKGKIDVTVGV KIHRGYKTKYKIMPLELKTGKESNSIEHRSQVVLYTLLSQERRADPEAGLLLYLKTGQMYPVPANHLDKRELLKLRNQMAFSLFHRISKSATRQ KTQLASLPQIIEEEKTCKYCSQIGNCALYSRAVEQQMDCSSVPIVMLPKIEEETQHLKQTHLEYFSLWCLMLTLESQSKDNKKNHQNIWLMPAS EMEKSGSCIGNLIRMEHVKIVCDGQYLHNFQCKHGAIPVTNLMAGDRVIVSGEERSLFALSRGYVKEINMTTVTCLLDRNLSVLPESTLFRLDQ EEKNCDIDTPLGNLSKLMENTFVSKKLRDLIIDFREPQFISYLSSVLPHDAKDTVACILKGLNKPQRQAMKKVLLSKDYTLIVGMPGTGKTTTI CTLVRILYACGFSVLLTSYTHSAVDNILLKLAKFKIGFLRLGQIQKVHPAIQQFTEQEICRSKSIKSLALLEELYNSQLIVATTCMGINHPIFS RKIFDFCIVDEASQISQPICLGPLFFSRRFVLVGDHQQLPPLVLNREARALGMSESLFKRLEQNKSAVVQLTVQYRMNSKIMSLSNKLIYEGKL ECGSDKVANAVINLRHFKDVKLELEFYADYSDNPWLMGVFEPNNPVCFLNTDKVPAPEQVEKGGVSNVTEAKLIVFLTSIFVKAGCSPSDIGII APYRQQLKIINDLLARSIGMVEVNTVDKYQGRDKSIVLVSFVRSNKDGTVGELLKDWRRLNVAITRAKHKLILLGCVPSLNCYPPLEKLLNHLN SEKLIIDLPSREHESLCHILGDFQRE Ezh2 >sp|Q15910|EZH2_HUMAN Histone-lysine N-methyltransferase EZH2 OS = Homo sapiens GN = EZH2 PE = 1 SV = 2 (SEQ ID NO: 2282) MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQ VIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDD DGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFH TLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESK DTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFR VKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKA QCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVY DKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIP >sp|Q15910-2|EZH2_HUMAN Isoform 2 of Histone-lysine N-methyltransferase EZH2 OS = Homo sapiens GN = EZH2 (SEQ ID NO: 2283) MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQ VIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDD DGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFH TLFCRRCFKYDCFLHRKCNYSFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTIN VLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQ VYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPG CRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADR RGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIP >sp|Q15910-3|EZH2_HUMAN Isoform 3 of Histone-lysine N-methyltransferase EZH2 OS = Homo sapiens GN = EZH2 (SEQ ID NO: 2284) MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTREVEDETVLHNIPY MGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAI SSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCG PQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQ TPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLK KDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKN CSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLENLNNDFVVDATRKGNKIRFANHSVNPNCYA KVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIP HDAC1 >sp|Q13547|HDAC1_HUMAN Histone deacetylase 1 OS = Homo sapiens GN = HDAC1 PE = 1 SV = 1 (SEQ ID NO: 2285) MAQTQGTRRKVCYYYDGDVGNYYYGQGHPMKPHRIRMTHNLLLNYGLYRKMEIYRPHKANAEEMTKYHSDDYIKFLRSIRPDNMSEYSKQMQRF NVGEDCPVFDGLFEFCQLSTGGSVASAVKLNKQQTDIAVNWAGGLHHAKKSEASGFCYVNDIVLAILELLKYHQRVLYIDIDIHHGDGVEEAFY TTDRVMTVSFHKYGEYFPGTGDLRDIGAGKGKYYAVNYPLRDGIDDESYEAIFKPVMSKVMEMFQPSAVVLQCGSDSLSGDRLGCFNLTIKGHA KCVEFVKSFNLPMLMLGGGGYTIRNVARCWTYETAVALDTEIPNELPYNDYFEYFGPDFKLHISPSNMTNQNTNEYLEKIKQRLFENLRMLPHA PGVQMQAIPEDAIPEESGDEDEDDPDKRISICSSDKRIACEEEFSDSEEEGEGGRKNSSNFKKAKRVKTEDEKEKDPEEKKEVTEEEKTKEEKP EAKGVKEEVKLA HDAC2 (SEQ ID NO: 2286) MAYSQGGGKKKVCYYYDGDIGNYYYGQGHPMKPHRIRMTHNLLLNYGLYRKMETYRPHKATAEEMTKYHSDEYIKFLRSIRPDNMSEYSKQMQR FNVGEDCPVFDGLFEFCQLSTGGSVAGAVKLNRQQTDMAVNWAGGLHHAKKSEASGFCYVNDIVLAILELLKYHQRVLYIDIDIHHGDGVEEAF YTTDRVMTVSFHKYGEYFPGTGDLRDIGAGKGKYYAVNFPMRDGIDDESYGQIFKPIISKVMEMYQPSAVVLQCGADSLSGDRLGCFNLTVKGH AKCVEVVKTFNLPLLMLGGGGYTIRNVARCWTYETAVALDCEIPNELPYNDYFEYFGPDFKLHISPSNMTNQNTPEYMEKIKQRLFENLRMLPH APGVQMQAIPEDAVHEDSGDEDGEDPDKRISIRASDKRIACDEEFSDSEDEGEGGRRNVADHKKGAKKARIEEDKKETEDKKTDVKEEDKSKDN SGEKTDTKGTKSEQLSNP KDM4A/JMJD2A >sp|O75164|KDM4A_HUMAN Lysine-specific demethylase 4A OS = Homo sapiens GN = KDM4A PE = 1 SV = 2 (SEQ ID NO: 2287) MASESETLNPSARIMTFYPTMEEFRNFSRYIAYIESQGAHRAGLAKVVPPKEWKPRASYDDIDDLVIPAPIQQLVTGQSGLFTQYNIQKKAMTV REFRKIANSDKYCTPRYSEFEELERKYWKNLTFNPPIYGADVNGTLYEKHVDEWNIGRLRTILDLVEKESGITIEGVNTPYLYFGMWKTSFAWH TEDMDLYSINYLHFGEPKSWYSVPPEHGKRLERLAKGFFPGSAQSCEAFLRHKMTLISPLMLKKYGIPFDKVTQEAGEFMITFPYGYHAGFNHG FNCAESTNFATRRWIEYGKQAVLCSCRKDMVKISMDVFVRKFQPERYKLWKAGKDNTVIDHTLPTPEAAEFLKESELPPRAGNEEECPEEDMEG VEDGEEGDLKTSLAKHRIGTKRHRVCLEIPQEVSQSELFPKEDLSSEQYEMTECPAALAPVRPTHSSVRQVEDGLTFPDYSDSTEVKFEELKNV KLEEEDEEEEQAAAALDLSVNPASVGGRLVFSGSKKKSSSSLGSGSSRDSISSDSETSEPLSCRAQGQTGVLTVHSYAKGDGRVTVGEPCTRKK GSAARSFSERELAEVADEYMFSLEENKKSKGRRQPLSKLPRHHPLVLQECVSDDETSEQLTPEEEAEETEAWAKPLSQLWQNRPPNFEAEKEFN ETMAQQAPHCAVCMIFQTYHQVEFGGFNQNCGNASDLAPQKQRTKPLIPEMCFTSTGCSTDINLSTPYLEEDGTSILVSCKKCSVRVHASCYGV PPAKASEDWMCSRCSANALEEDCCLCSLRGGALQRANDDRWVHVSCAVAILEARFVNIAERSPVDVSKIPLPRFKLKCIFCKKRRKRTAGCCVQ CSHGRCPTAFHVSCAQAAGVMMQPDDWPFVVFITCFRHKIPNLERAKGALQSITAGQKVISKHKNGRFYQCEVVRLTTETFYEVNFDDGSFSDN LYPEDIVSQDCLQFGPPAEGEVVQVRWTDGQVYGAKFVASHPIQMYQVEFEDGSQLVVKRDDVYTLDEELPKRVKSRLSVASDMRFNEIFTEKE VKQEKKRQRVINSRYREDYIEPALYRAIME >sp|O75164-2|KDM4A_HUMAN Isoform 2 of Lysine-specific demethylase 4A OS = Homo sapiens GN = KDM4A (SEQ ID NO: 2288) MFSLEENKKSKGRRQPLSKLPRHHPLVLQECVSDDETSEQLTPEEEAEETEAWAKPLSQLWQNRPPNFEAEKEFNETMAQQAPHCAVCMIFQTY HQVEFGGFNQNCGNASDLAPQKQRTKPLIPEMCFTSTGCSTDINLSTPYLEEDGTSILVSCKKCSVRVHASCYGVPPAKASEDWMCSRCSANAL EEDCCLCSLRGGALQRANDDRWVHVSCAVAILEARFVNIAERSPVDVSKIPLPRFKLKCIFCKKRRKRTAGCCVQCSHGRCPTAFHVSCAQAAG VMMQPDDWPFVVFITCFRHKIPNLERAKGALQSITAGQKVISKHKNGRFYQCEVVRLTTETFYEVNFDDGSFSDNLYPEDIVSQDCLQFGPPAE GEVVQVRWTDGQVYGAKFVASHPIQMYQVEFEDGSQLVVKRDDVYTLDEELPKRVKSRLSVASDMRFNEIFTEKEVKQEKKRQRVINSRYREDY IEPALYRAIME

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Headings, including numeric and alphabetical headings and subheadings, are for organization and presentation and are not intended to be limiting.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. 

1. A Cas9 system comprising: an HDR-enhancer molecule, wherein the enhancer molecule is not an inhibitor of DNA-PK or an inhibitor of Ligase IV, a Cas9 molecule, and a gRNA molecule, wherein the gRNA molecule is capable of targeting the Cas9 molecule to a target nucleic acid.
 2. The Cas9 system of claim 1, wherein the HDR-enhancer molecule is an HDR-enhancing gRNA, an siRNA, a peptide, an antibody, an miRNA, an antiMiR, or a small molecule.
 3. The Cas9 system of claim 1, further comprising a second gRNA molecule, wherein the second gRNA molecule is capable of targeting the Cas9 molecule to the target nucleic acid.
 4. A Cas9 system comprising: a Cas9 nickase molecule, a gRNA molecule, wherein the gRNA molecule is capable of targeting the Cas9 nickase molecule to a target nucleic acid, and a second gRNA molecule, wherein the second gRNA molecule is capable of targeting the Cas9 nickase molecule to the target nucleic acid, wherein the gRNA molecule and the second gRNA molecule are designed to be oriented on the target nucleic acid such that protospacer adjacent motifs (PAMs) are facing out, wherein the gRNA molecule will position the Cas9 nickase molecule to make a single-strand break in the target nucleic acid which results a 5′ overhang in the target nucleic acid.
 5. The Cas9 system of claim 1, further comprising a template nucleic acid. 6.-9. (canceled)
 10. The Cas9 system of claim 1, wherein the HDR-enhancer is a down-regulator of HR, a down-regulator of canonical NHEJ, a down-regulator of alt-NHEJ, a down-regulator of an antirecombinant factor, a down-regulator of SSA, a down-regulator of SSBR, a down-regulator of MMR, a chromatin modification agent, a cell cycle arrest compound, an agent capable of promoting resection at a double strand break, a down-regulator of SD-MMEJ, an up-regulator of HDR, or a down-regulator of blunt EJ. 11.-62. (canceled)
 63. The Cas9 system of claim 1, which further comprises at least one additional HDR-enhancer molecule. 64.-66. (canceled)
 67. The Cas9 system of claim 1, wherein the Cas9 molecule is fused to a transcription activator or a transcription repressor.
 68. The Cas9 system of claim 1, wherein the Cas9 molecule is an enzymatically inactive Cas9 molecule (eiCas9) or an enzymatically active Cas9 (eaCas9) molecule. 69.-74. (canceled)
 75. The Cas9 system of claim 1, further comprising a second Cas9 molecule. 76.-93. (canceled)
 94. The Cas9 system of claim 1 further comprising a cell cycle arrest compound. 95.-96. (canceled)
 97. A Cas9 system comprising: a Cas9 nickase molecule, a gRNA molecule, wherein the gRNA molecule is capable of targeting the Cas9 nickase molecule to a target nucleic acid, and a second gRNA molecule, wherein the second gRNA molecule is capable of targeting the Cas9 nickase molecule to the target nucleic acid, wherein the gRNA molecule and the second gRNA molecule are designed to be oriented on the target nucleic acid such that protospacer adjacent motifs (PAMs) are facing out, wherein the gRNA molecule will position the Cas9 nickase molecule to make a single-strand break in the target nucleic acid which results a 3′ overhang in the target nucleic acid.
 98. A cell comprising the Cas9 system of claim
 1. 99. A composition comprising the Cas9 system of claim
 1. 100. A pharmaceutical composition comprising the composition of claim 99 and a pharmaceutically acceptable carrier.
 101. A kit comprising the Cas9 system of claim
 1. 102. (canceled)
 103. A vector comprising the Cas9 system of claim
 1. 104.-105. (canceled)
 106. A reaction mixture comprising the cell of claim 98, and a solution.
 107. (canceled)
 108. A method of altering the structure of a cell comprising contacting the cell with: a Cas9 system of claim 1, under conditions that allow for alteration of the structure of the cell, thereby altering the structure of the cell.
 109. (canceled)
 110. A method of treating a subject by altering the structure of a cell in the subject, comprising contacting the cell with: a Cas9 system of claim 1 under conditions that allow for alteration of the structure of the cell, thereby treating the subject by altering the structure of the cell in the subject.
 111. A method of promoting DNA repair of a break in a nucleic acid in a cell via an HDR pathway, the method comprising contacting the cell with: a Cas9 system of claim 1 under conditions that allow for repair of the break in the nucleic acid in the cell via an HDR pathway.
 112. A method of promoting DNA repair of a double strand break or two single strand breaks in a target nucleic acid in a cell by an HDR pathway, the method comprising contacting the cell with a gRNA molecule, a Cas9 molecule, and a second gRNA molecule, wherein the gRNA molecule and the second gRNA molecule are oriented on the target nucleic acid such that protospacer adjacent motifs (PAMs) are facing out, wherein the Cas9 nickase molecule cuts the target nucleic acid, resulting in a first 5′ overhang and a second 5′ overhang, thereby promoting DNA repair of the double strand break or the two single strand breaks in the target nucleic acid in the cell via an HDR pathway. 113.-128. (canceled)
 129. The method of claim 110, wherein the subject has a disorder that is caused by a mutation in the target nucleic acid. 130.-136. (canceled)
 137. A method of producing the Cas9 system of claim 1, the method comprising providing and admixing the gRNA molecule, the Cas9 molecule, and the HDR-enhancer molecule.
 138. (canceled) 